RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Regulation of growth cone actin filaments by guidance cues

The motile behaviors of growth cones at the ends of elongating axons determine pathways of axonal connections in developing nervous systems. Growth cones express receptors for molecular guidance cues in the local environment, and receptor-guidance cue binding initiates cytoplasmic signaling that regulates the cytoskeleton to control growth cone advance, turning, and branching behaviors. The dynamic actin filaments of growth cones are frequently targets of this regulatory signaling. Rho GTPases are key mediators of signaling by guidance cues, although much remains to be learned about how growth cone responses are orchestrated by Rho GTPase signaling to change the dynamics of polymerization, transport, and disassembly of actin filaments. Binding of neurotrophins to Trk and p75 receptors on growth cones triggers changes in actin filament dynamics to regulate several aspects of growth cone behaviors. Activation of Trk receptors mediates local accumulation of actin filaments, while neurotrophin binding to p75 triggers local decrease in RhoA signaling that promotes lengthening of filopodia. Semaphorin IIIA and ephrin-A2 are guidance cues that trigger avoidance or repulsion of certain growth cones, and in vitro responses to these proteins include growth cone collapse. Dynamic changes in the activities of Rho GTPases appear to mediate responses to these cues, although it remains unclear what the changes are in actin filament distribution and dynamic reorganization that result in growth cone collapse. Growth cones in vivo simultaneously encounter positive and negative guidance cues, and thus, growth cone behaviors during axonal pathfinding reflect the complex integration of multiple signaling activities.     

Hepatocyte growth factor and its receptor are required for malaria infection

Plasmodium, the causative agent of malaria, must first infect hepatocytes to initiate a mammalian infection. Sporozoites migrate through several hepatocytes, by breaching their plasma membranes, before infection is finally established in one of them. Here we show that wounding of hepatocytes by sporozoite migration induces the secretion of hepatocyte growth factor (HGF), which renders hepatocytes susceptible to infection. Infection depends on activation of the HGF receptor, MET, by secreted HGF. The malaria parasite exploits MET not as a primary binding site, but as a mediator of signals that make the host cell susceptible to infection. HGF/MET signaling induces rearrangements of the host-cell actin cytoskeleton that are required for the early development of the parasites within hepatocytes. Our findings identify HGF and MET as potential targets for new approaches to malaria prevention.     

AnkyrinG is required for maintenance of the axon initial segment and neuronal polarity

The axon initial segment (AIS) functions as both a physiological and physical bridge between somatodendritic and axonal domains. Given its unique molecular composition, location, and physiology, the AIS is thought to maintain neuronal polarity. To identify the molecular basis of this AIS property, we used adenovirus-mediated RNA interference to silence AIS protein expression in polarized neurons. Some AIS proteins are remarkably stable with half-lives of at least 2 wk. However, silencing the expression of the cytoskeletal scaffold ankyrinG (ankG) dismantles the AIS and causes axons to acquire the molecular characteristics of dendrites. Both cytoplasmic- and membrane-associated proteins, which are normally restricted to somatodendritic domains, redistribute into the former axon. Furthermore, spines and postsynaptic densities of excitatory synapses assemble on former axons. Our results demonstrate that the loss of ankG causes axons to acquire the molecular characteristics of dendrites; thus, ankG is required for the maintenance of neuronal polarity and molecular organization of the AIS.     

Electrical activity modulates growth cone guidance by diffusible factors

Brief periods of electrical stimulation of cultured Xenopus spinal neurons resulted in a marked alteration in the turning responses of the growth cone induced by gradients of attractive or repulsive guidance cues. Netrin-1-induced attraction was enhanced, and the repulsion induced by myelin-associated glycoprotein (MAG) or myelin membrane fragments was converted to attraction. The effect required the presence of extracellular Ca(2+) during electrical stimulation and appeared to be mediated by an elevation of both cytoplasmic Ca(2+) and cAMP. Thus, electrical activity may influence the axonal path finding of developing neurons, and intermittent electrical stimulation may be effective in promoting nerve regeneration after injury.     

Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast

Formation and maintenance of specialized plasma membrane domains are crucial for many biological processes, such as cell polarization and signaling. During isotropic bud growth, the yeast cell periphery is divided into two domains: the bud surface, an active site of exocytosis and growth, and the relatively quiescent surface of the mother cell. We found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth. Furthermore, we found that septins were required for proper regulation of actin patch stability; septin-defective cells permitted to enter isotropic growth lost actin and growth polarity. We propose that septins maintain cell polarity by specifying a boundary between cortical domains.     

Membrane tension, myosin force, and actin turnover maintain actin treadmill in the nerve growth cone

A growth cone is a motile structure at the tips of axons that is driven by the actin network and guides axon extension. Low actin adhesion to the substrate creates a stationary actin treadmill that allows leading-edge protrusion when adhesion increases in response to guidance cues. We use experimental measurements in the Aplysia bag growth cone to develop and constrain a simple mechanical model of the actin treadmill. We show that actin retrograde flow is primarily generated by myosin contractile forces, but when myosin is inhibited, leading-edge membrane tension increases and drives the flow. By comparing predictions of the model with previous experimental measurements, we demonstrate that lamellipodial and filopodial filament breaking contribute equally to the resistance to the flow. The fully constrained model clarifies the role of actin turnover in the mechanical balance driving the actin treadmill and reproduces the recent experimental observation that inhibition of actin depolymerization causes retrograde flow to slow exponentially with time. We estimate forces in the actin treadmill, and we demonstrate that measured G-actin distributions are consistent with the existence of a forward-directed fluid flow that transports G-actin to the leading edge.     

Epidermal growth factor-mediated transient phosphorylation and membrane localization of myosin II-B are required for efficient chemotaxis

Epidermal growth factor (EGF) stimulation of prostate metastatic tumor cells results in transient phosphorylation and cellular localization of non-muscle myosin heavy chain II-B (NMHC II-B) with kinetics similar to those seen in chemotaxis. We demonstrate that expression of 18- and 72-kDa fragments derived from the NMHC II-B C terminus that contain EGF-dependent NMHC II-B phosphorylation sites serve as dominant-negative mutations for EGF-dependent NMHC II-B phosphorylation and localization. Both fragments inhibited the EGF-dependent phosphorylation by competing with NMHC II-B on the myosin heavy chain kinase. However, only expression of the 72-kDa fragment resulted in cells with abnormalities in cell shape, focal adhesions, and chemotaxis. We found that the 72-kDa (but not 18-kDa) fragment is capable of self-assembly. To our knowledge, these results provide the first strong evidence that EGF-dependent NMHC II-B phosphorylation is required for the cellular localization of NMHC II-B and that NMHC II-B is required for normal cell attachment and for chemotactic response.     

Phospholipase C-gamma and phosphoinositide 3-kinase mediate cytoplasmic signaling in nerve growth cone guidance.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.     

Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro

The role(s) of one family of polypeptide growth factors in a developing organ system was examined. Renal anlagen (metanephroi) were surgically removed from 13-d-old rat embryos and grown in organ culture for up to 6 d. Over this period of time when placed in serum-free defined media, the metanephroi increased in size and morphologic complexity. Messenger RNAs for both insulin-like growth factors (IGFs), IGF I and IGF II, were present in the metanephroi. Immunoreactive IGF I and IGF II were produced by the renal anlagen and released into culture media. Levels were relatively constant during the 6 d in culture and averaged 3.5 X 10(-9) M IGF I and 8.3 X 10(-9) M IGF II in media removed from metanephroi after contact for 24 h. IGF binding protein activity was not detected in culture media. Growth and development of metanephroi in vitro was prevented by the addition of anti-IGF I or anti-IGF II antibodies to organ cultures. IGF II produced by metanephroi was active in an IGF II biological assay system and addition of anti-IGF II receptor antibodies to organ cultures prevented growth and development, consistent with the action of IGF II in metanephroi being mediated via the IGF II receptor. The data demonstrate production of both IGF I and IGF II by developing rat metanephroi in organ culture. Each of these peptides is necessary for growth and development of the renal anlage to take place in vitro. Our findings suggest that both IGF I and IGF II are produced within the developing metanephros in vivo and promote renal organogenesis.     

Signal transduction underlying growth cone guidance by diffusible factors.

Many diffusible axon guidance cues and their receptors have been identified recently. These cues are often found to be bifunctional, acting as attractants or repellents under different circumstances. Studies of cytoplasmic signaling mechanisms have led to the notion that the response of a growth cone to a particular guidance cue depends on the internal state of the neuron, which, in turn, is under the influence of other coincident signals received by the neuron. Furthermore, many diffusible guidance cues appear to share common cytoplasmic signaling pathways.     

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.     

Gradients and growth cone guidance of grasshopper neurons.

The generation of a functional nervous system is dependent on precise pathfinding of axons during development. This pathfinding is directed by the distribution of local and long-range guidance cues, the latter of which are believed to be distributed in gradients. Gradients of guidance cues have been associated with growth cone function for over a hundred years. However, little is known about the mechanisms used by growth cones to respond to these gradients, in part owing to the lack of identifiable gradients in vivo. In the developing grasshopper limb, two gradients of the semaphorin Sema-2a are necessary for correct neuronal pathfinding in vivo. The gradients are found in regions where growth cones make critical steering decisions. Observations of different growth cone behaviors associated with these gradients have provided some insights into how growth cones respond to them. Growth cones appear to respond more faithfully to changes in concentration, rather than absolute levels, of Sema-2a expression, whereas the absolute levels may regulate growth cone size.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial targeting of type II protein kinase A to filopodia mediates the regulation of growth cone guidance by cAMP

The second messenger cyclic adenosine monophosphate (cAMP) plays a pivotal role in axonal growth and guidance, but its downstream mechanisms remain elusive. In this study, we report that type II protein kinase A (PKA) is highly enriched in growth cone filopodia, and this spatial localization enables the coupling of cAMP signaling to its specific effectors to regulate guidance responses. Disrupting the localization of PKA to filopodia impairs cAMP-mediated growth cone attraction and prevents the switching of repulsive responses to attraction by elevated cAMP. Our data further show that PKA targets protein phosphatase-1 (PP1) through the phosphorylation of a regulatory protein inhibitor-1 (I-1) to promote growth cone attraction. Finally, we find that I-1 and PP1 mediate growth cone repulsion induced by myelin-associated glycoprotein. These findings demonstrate that the spatial localization of type II PKA to growth cone filopodia plays an important role in the regulation of growth cone motility and guidance by cAMP.     

Axon guidance: opposing EPHects in the growth cone

Cells communicate with other cells via (trans) interaction between membrane-linked ephrins and Eph receptors. In this issue of Cell, Pfaff and colleagues (Marquardt et al., 2005) demonstrate that coexpressed ephrin-As and Ephs do not interact in cis but rather segregate into separate membrane domains, from which they signal opposing effects during motor axon guidance.     

Semaphorins: mediators of repulsive growth cone guidance

During development, neuronal growth cones encounter a variety of guidance cues while mediating axon path finding, target recognition and synapse formation. It is clear that repulsive guidance mechanisms play an essential role in these processes. The semaphorin gene family, which is conserved from invertebrates to mammals, includes members that mediate repulsive guidance. Molecular and cellular analysis of this gene family is providing insight into how inhibitory cues function during neurodevelopment.