The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones

Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.     

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.     

Semaphorin 3A inhibits ERM protein phosphorylation in growth cone filopodia through inactivation of PI3K

Ezrin-radixin-moesin (ERM) proteins are involved in the linkage of membranes to theactin filament (F-actin) cytoskeleton. Phosphorylation of the C-terminus activates the F-actin binding domain of ERM proteins by preventing the action of an autoinhibitory domain. In this study, we investigated whether a growth cone collapsing signal, semaphorin 3A (Sema3A), alters the state of ERM C-terminus phosphorylation. In the growth cones of dorsal root ganglion axons, phosphorylated ERM proteins localize to filopodia. We report that Sema3A inhibits ERM protein phosphorylation in growth cone filopodia. Significantly, Sema3A decreased ERM phosphorylation prior to the onset of growth cone collapse. Over-expression of the F-actin binding fragment of ERM proteins, which competes with endogenous ERM proteins for binding to F-actin, inhibited filopodial initiation and dynamics. Sema3A has been previously shown to inhibit phosphoinositide 3-kinase (PI3K) activity. Inhibition of PI3K resulted in the loss of phosphorylated ERM proteins from growth cone filopodia, and treatment with a PI3K activating peptide blocked the effects of Sema3A on ERM phosphorylation. Collectively, these observations demonstrate that inactivation of PI3K in response to Sema3A results in decreased phosphorylation of ERM proteins in filopodia thereby contributing to growth cone collapse.     

Regulation of actomyosin contractility by PI3K in sensory axons

Phosphatidylinositol 3-kinase (PI3K) activity is known to be required for the extension of embryonic sensory axons. Inhibition of PI3K has also been shown to mediate axon retraction and growth cone collapse in response to semaphorin 3A. However, the effects of inhibiting PI3K on the neuronal cytoskeleton are not well characterized. We have previously reported that semaphorin 3A-induced axon retraction involves activation of myosin II, the formation of an intra-axonal F-actin bundle cytoskeleton, and blocks the formation of F-actin patches that serve as precursors to filopodial formation in axons. We now report that inhibition of PI3K results in activation of myosin II in axons. Inhibition of myosin II activity, or its upstream regulatory kinase RhoA-kinase, blocked axon retraction induced by inhibition of PI3K. In addition, inhibition of PI3K also induced intra-axonal F-actin bundles, which likely serve as a substratum for myosin II-based force generation during axon retraction. In axons, filopodia are formed from axonal F-actin patch precursors. Analysis of axonal F-actin patch formation in eYFP-actin expressing neurons revealed that inhibition of PI3K blocked formation of axonal F-actin patches, and thus filopodial formation. These data provide insights into the regulation of the neuronal cytoskeleton by PI3K and are consistent with the notion that decreased levels of PI3K activity mediate axon retraction and growth cone collapse in response to semaphorin 3A.     

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.     

Distribution of GAP-43, β-III tubulin and F-actin in developing and regenerating axons and their growth cones in vitro, following neurotrophin treatment

Brain derived neurotrophic factor (BDNF) when added to explant cultures of both embryonic and adult retinal ganglion cell (RGC) axons exerted a marked effect on their growth cone size and complexity and also on the intensity of GAP-43, ß-III tubulin and F-actin immunoreaction product in their axons. GAP-43 was distributed in axons, lamellipodia, and filopodia whereas ß-III tubulin was distributed along the length of developing and adult regenerating axons and also in the C-domain of their growth cones. BDNF-treated developing RGC growth cones were larger and displayed increased numbers of GAP-43 and microtubule-containing branches. Although filopodia and lamellipodia were lost from both developing and adult RGC growth cones following trkB-IgG treatment, the intensity of the immunoreaction product of all these molecules was reduced and trkB-IgGs had no effect on the axonal distribution of ß-III tubulin and GAP-43. BDNF-treated growth cones also displayed increased numbers of F-actin containing filopodia and axonal protrusions. This study demonstrates, for the first time, that trkB-IgG treatment causes the loss of F-actin in the P-domain of growth cone tips in developing and regenerating RGC axons. Although microtubules and F-actin domains normally remained distinct in cultured growth cones, ß-III tubulin and F-actin overlapped within the growth cone C-domain, and within axonal protrusions of adult RGC axons, under higher concentrations of BDNF. The collapse of RGC growth cones appeared to correlate with the loss of F-actin. In vitro, trkB signalling may therefore be involved in the maintenance and stabilisation of RGC axons, by influencing F-actin polymerisation, stabilisation and distribution.     

Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions

Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.     

Kinase requirement for retinal growth cone motility

Since cytoplasmic Ca²⁺ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantifed over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1–1 μM) and chelerythrin (up to 50 μM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 μM), trifluoperazine (100 μM), and CGS9343B (50 μM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 μM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 μM), produced no effects but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3–5 μM) and ML-9 (5–10 μM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 μM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts. 1994 John Wiley & Sons, Inc.     

The 2-arachidonoylglycerol effect on myosin light chain phosphorylation in human platelets

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B₂ formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.     

Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.     

Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.     

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.     

Myosin light chain phosphorylation-dependent modulation of volume-regulated anion channels in macrovascular endothelium

The Rho/Rho-associated kinase (ROK) pathway has been shown to modulate volume-regulated anion channels (VRAC) in cultured calf pulmonary artery endothelial (CPAE) cells. Since Rho/ROK can increase myosin light chain phosphorylation, we have now studied the effects of inhibitors of myosin light chain kinase (MLCK) or myosin light chain phosphatase (MLCP) on VRAC in CPAE. Application of ML-9, an MLCK inhibitor, inhibited VRAC, both when applied extracellularly or when dialyzed into the cell. A similar inhibitory effect was obtained by dialyzing the cells with AV25, a specific MLCK inhibitory peptide. Conversely, NIPP1(191-210), an MLCP inhibitory peptide, potentiated the activation of VRAC by a 25% hypotonic stimulus. These data indicate that activation of VRAC is modulated by MLC phosphorylation.     

Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow-a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.     

Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.     

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

A subset of signal transduction pathways is required for hippocampal growth cone collapse induced by ephrin-A5

The Eph family tyrosine kinase receptors and their ligands, ephrins, play key roles in a wide variety of physiological and pathological processes including tissue patterning, angiogenesis, bone development, carcinogenesis, axon guidance, and neural plasticity. However, the signaling mechanisms underlying these diverse functions of Eph receptors have not been well understood. In this study, effects of Eph receptor activation on several important signal transduction pathways are examined. In addition, the roles of these pathways in ephrin-A5-induced growth cone collapse were assessed with a combination of biochemical analyses, pharmacological inhibition, and overexpression of dominant-negative and constitutively active mutants. These analyses showed that ephrin-A5 inhibits Erk activity but activates c-Jun N-terminal kinase. However, regulation of these two pathways is not required for ephrin-A5-induced growth cone collapse in hippocampal neurons. Artificial Erk activation by expression of constitutively active Mek1 and B-Raf failed to block ephrin-A5 effects on growth cones, and inhibitors of the Erk pathway also failed to inhibit collapse by ephrin-A5. Inhibition of JNK had no effects on ephrin-A5-induced growth cone collapse either. In addition, inhibitors to PKA and PI3-K showed no effects on ephrin-A5-induced growth cone collapse. However, pharmacological blockade of phosphotyrosine phosphatase activity, the Src family kinases, cGMP-dependent protein kinase, and myosin light chain kinase significantly inhibited ephrin-A5-induced growth cone collapse. These observations indicate that only a subset of signal transduction pathways is required for ephrin-A5-induced growth cone collapse.     

Regulation of Cell Motility by Mitogen-activated Protein Kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Retrograde flow and myosin II activity within the leading cell edge deliver F-actin to the lamella to seed the formation of graded polarity actomyosin II filament bundles in migrating fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.