BIOLOGICAL OVERVIEW

Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. The actin-depolymerizing and actin-severing protein factor cofilin, encoded by twinstar, is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rho-associated kinase (Rok, also named Rho kinase or ROCK), or p21-activated kinase (Pak), respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection (Ng, 2004).

From yeast to mammals, cofilin plays an essential morphogenetic role by promoting the rapid turnover of actin filaments through severing filamentous actin (F-actin) and depolymerizing actin filaments from the pointed ends (Bamburg, 1999). Mutations in the twinstar (tsr) gene, which encodes the unique Drosophila homolog of cofilin, result in neuroblast proliferation, spermatogenesis, and defects in epithelial morphogenesis (Gunsalus, 1995; Chen, 2001). Drosophila cofilin was first identified in a screen for genes that induce aberrant cell shapes in fission yeast (Edwards, 1994). In mammalian cells, cofilin activity is inhibited by phosphorylation at serine 3, which is mediated by LIM kinase (LIMK). LIMK is activated through phosphorylation by Pak or Rok effector kinases for Rac/Cdc42 or Rho, respectively. In Drosophila, one LIMK gene has been found that can also phosphorylate cofilin at serine 3 (LIMK1-Flybase) (Ohashi, 2000), but how LIMK1 is regulated is unknown. Cofilin is dephosphorylated by Slingshotphosphatase (Ssh) (Niwa, 2002). Drosophila ssh mutants exhibit defects in epithelial morphogenesis that are characterized by high levels of F-actin and cofilin phosphorylation, suggesting that Ssh regulates actin dynamics through cofilin dephosphorylation (Ng, 2004 and references therein).

The function and regulation of cofilin in neuronal morphogenesis in vivo has not been reported. Loss of cofilin is shown to result in severe axon growth defects in Drosophila neurons. Cofilin function is positively regulated by Ssh phosphatase and negatively regulated by LIMK during axon growth. Cofilin dephosphorylation appears to be the major function of Ssh, since axon growth defects in ssh mutant neurons are suppressed by expressing active forms of cofilin. Genetic evidence is provided that both the Rho-Rok and the Rac/Cdc42-Pak pathways positively regulate Drosophila LIMK. Importantly, while the LIMK pathway acts to inhibit axon growth, Rac also signals through a Pak-independent pathway that acts antagonistically to LIMK to promote axon growth. Furthermore, two distinct RacGEFs appear to be selectively engaged to regulate these different pathways. These genetic results indicate that multiple Rho GTPase signaling pathways converge on a common downstream target, cofilin, to regulate axon growth. At the same time, Rho GTPases also regulate divergent downstream pathways that act in an antagonistic fashion to coordinate growth cone motility (Ng, 2004).

Using Drosophila mushroom body neurons as a genetic model, several signaling pathways through which Rho GTPases regulate axon growth in vivo were examined. Several new insights were obtained from these genetic analyses. Actin polymerization at the cellular leading edge is generally thought to provide the driving force for membrane protrusions such as lamellar extension in migrating cells or filopodia and lamellipodia extensions for neuronal growth cone advance. Cofilin was found to be essential for axon growth in vivo. Since cofilin has both pointed-end depolymerization activity and F-actin severing activity, there are at least two possible explanations, on the basis of biochemical and cell biological studies in other cell types, for its essential role in axon growth (e.g., Carlier, 1997; Svitkina, 1999; Dawe, 2003; Ghosh, 2004). (1) Actin polymerization at the leading edge requires a constant supply of monomeric actin subunits derived from depolymerization at the pointed end. (2) The severing activity of cofilin allows generation of free barbed ends as templates for actin polymerization. While the contributions of either of the above processes to axon growth have not been ruled out, it was found that loss of cofilin does not simply result in a lack of filopodia or lamellipodia. Instead, the overabundance of filopodia- and lamellipodia-like structures retained in cofilin mutant axons suggests a third possibility: growth cone advance is inhibited when filopodia/lamellipodia cannot be disassembled upon the loss of cofilin activity (Ng, 2004).

How is cofilin activity regulated during axon growth? LIM Kinase and Ssh Phosphatase are known to be key regulators of cofilin in axon growth. A number of recent studies have addressed the role of cofilin phosphorylation by LIM-kinase and Ssh phosphatase in cultured neurons. For example, overexpression of active forms of cofilin in rat cortical neurons results in an increase in both the number of filopodia and the degree of neurite extension (Meberg, 2000). Overexpression of active forms of LIMK in chick dorsal root ganglion neurons represses growth cone motility and neurite extension. The LIMK effects are likely to be mediated through cofilin, since cotransfection of either mammalian Ssh or the S3A form of cofilin suppresses the LIMK effects (Endo, 2003). Cofilin phosphorylation by LIMK (Aizawa, 2001) is further implicated in semaphorin-mediated growth cone collapse (Ng, 2004).

In vivo study in Drosophila confirms and extends these in vitro studies in several ways. (1) Using a transgenic rescue assay, it was shown that neither phosphomimetic (S3E) nor nonphosphorylatable (S3A) cofilin or their combination can replace endogenous cofilin function. This suggests that cycles of cofilin phosphorylation ('inactivation') and dephosphorylation ('reactivation') are important during actin turnover to promote axon growth and that in vivo the factors that regulate cofilin phosphorylation must act in a delicate balance to optimize axon growth during development. (2) Loss of Ssh is shown to result in axon growth defects, and these defects can be suppressed by the expression of active cofilin, demonstrating that the major function of Ssh in regulating axon growth is cofilin dephosphorylation. (3) LIMK overexpression is shown to result in axon growth defects analogous to ssh, and this phenotype can be suppressed by the coexpression of Ssh or active cofilin. Taken together with existing biochemical data, these results firmly establish that regulation of cofilin phosphorylation by Ssh phosphatase and LIMK plays a pivotal role in regulating axon growth in vivo (Ng, 2004).

Although cofilin phosphorylation is essential for neuroblast proliferation, neither LIMK nor Ssh appears to be a key regulator. No cell proliferation defects were detected in ssh^-/- neuroblast clones or in LIMK1-overexpressing neurons, in contrast to tsr^-/- clones. It is unlikely that cell proliferation is less sensitive to the reduction of cofilin activity than is axon growth. On the contrary, neuroblast clones homozygous for a hypomorphic allele of tsr (tsr¹) have strong defects in cell proliferation, but no defects in axon growth. Taken together, these data suggest that cofilin phosphorylation during cell proliferation is regulated by a set of kinases/phosphatases different from those that regulate axon growth (Ng, 2004).

Genetic analyses show that Rho, Cdc42, and Rac all contribute to activation of the LIMK1 pathway, which leads to axon growth inhibition. However, previous cell biological data in vitro (Kozma, 1997) and loss-of-function mutant analysis in vivo (Lundquist 2001; Hakeda-Suzuki, 2002; Ng, 2002) indicate that Rac GTPases act to promote axon growth. How can one resolve these seemingly opposite effects of Rac GTPases? Several lines of evidence are provided that, in addition to activating LIMK1, Rac GTPases also act via a second pathway to promote axon growth. (1) Reduction of Rac GTPase activity can also enhance the LIMK1 overexpression phenotype, suggesting that Rac can act antagonistically to LIMK1 to promote axon growth. (2) Overexpressing Rac1 Y40C (a mutant with diminished binding to Pak) strongly suppresses the LIMK1 overexpression phenotype. Since Pak activation leads to axon growth inhibition and Pak^-/- neuroblast clones do not have axon growth defects, these data together suggest that the Rac pathway that counteracts the LIMK pathway is Pak independent, which is consistent with previous studies in which transgenically supplied Rac1 Y40C rescued axon growth in the absence of all endogenous Rac (Ng, 2002). (3) Two different RacGEFs have been shown to either enhance or suppress the LIMK pathway. This again suggests that different Rac signaling pathways act antagonistically to regulate axon growth (Ng, 2004).

Given the presence of these two Rac GTPase pathways (LIMK1--->Twinstar dependent and independent pathways), it is likely that, depending on the signaling context, Rac can either inhibit or promote axon growth. Indeed, Rac activation has been shown to either promote or inhibit axon growth in different systems (reviewed in Luo, 2000). In addition, both attractive and repulsive axon guidance cues can signal through Rac GTPases to mediate these opposite effects in vivo. It is proposed that one possible explanation for the above phenomena is that different cues selectively favor either the Pak/LIMK/cofilin phosphorylation pathway or the alternative axon growth-promoting pathway, resulting in different developmental outcomes. The finding that two RacGEFs (Trio and Still life) have opposite effects in modifying LIMK activity suggests that the selection of these pathways could be achieved by selectively engaging different GEFs. How RacGEFs selectively couple to different downstream effector pathways remains to be determined by future experiments (Ng, 2004).

Rac activation stimulates actin polymerization and leads to cell protrusions via lamellipodia formation. However, testing of several major classes of actin polymerization stimulators did not provide evidence that Rac promotes axon growth through the actin polymerization pathway. For instance, the SCAR-Arp2/3 pathway essential for de novo actin polymerization does not appear to contribute to MB axon growth in vivo. This is consistent with a recent study suggesting that the Arp2/3 pathway is also not essential for axon growth in cultured neurons. Although the anticapping protein Enabled is essential for MB axon growth, genetic interaction data argue against its participation in the axon growth-promoting pathway downstream of Rac. This is also consistent with previous genetic analysis in C. elegans, indicating that Rac (Ced-10) and Ena (Unc-34) act in parallel pathways downstream of the netrin receptor to promote axon growth. Another possibility for the axon growth-promoting pathway is that Rac counteracts the LIMK pathway by activating Ssh. Recent in vitro data suggest that Rac can act to dephosphorylate cofilin (Nagata-Ohashi, 2004), thereby promoting actin turnover. However, it is believed that the Rac-dependent axon growth pathway is unlikely to be via Ssh alone, given that, in the absence of ssh, Rac Y40C overexpression can still suppress the ssh growth phenotype. Since axon growth also requires the regulation of microtubule dynamics and vesicle trafficking, both of which are thought to be Rho GTPase dependent, it is proposed that the Rac-mediated axon growth-promoting pathway may involve these processes (Ng, 2004).

In summary, genetic analyses have begun to tease apart the complex signaling networks between Rho GTPases and the actin cytoskeleton in the context of axon growth in vivo. Rho GTPases act through convergent and divergent signaling pathways to regulate axon growth. In addition to cofilin regulation, analyses of other actin polymerization regulators in MB neurons have established the relationship between these signaling pathways and the regulation of axon growth. The pathways identified in this study provide a foundation for future investigations as to how extracellular cues direct growth cone signaling to precisely wire neural circuits in vivo (Ng, 2004).

Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin

The Drosophila neuromuscular junction (NMJ) is capable of rapidly budding new presynaptic varicosities over the course of minutes in response to elevated neuronal activity. Using live imaging of synaptic growth, this dynamic process was characterized, and it was demonstrated that rapid bouton budding requires retrograde bone morphogenic protein (BMP) signaling and local alteration in the presynaptic actin cytoskeleton. BMP acts during development to provide competence for rapid synaptic growth by regulating the levels of the Rho-type guanine nucleotide exchange factor Trio, a transcriptional output of BMP-Smad signaling. In a parallel pathway, it was found that the BMP type II receptor Wit signals through the effector protein LIM domain kinase 1 (Limk) to regulate bouton budding. Limk interfaces with structural plasticity by controlling the activity of the actin depolymerizing protein Cofilin. Expression of constitutively active or inactive Cofilin (Twinstar) in motor neurons demonstrates that increased Cofilin activity promotes rapid bouton formation in response to elevated synaptic activity. Correspondingly, the overexpression of Limk, which inhibits Cofilin, inhibits bouton budding. Live imaging of the presynaptic F-actin cytoskeleton reveals that activity-dependent bouton addition is accompanied by the formation of new F-actin puncta at sites of synaptic growth. Pharmacological disruption of actin turnover inhibits bouton budding, indicating that local changes in the actin cytoskeleton at pre-existing boutons precede new budding events. It is proposed that developmental BMP signaling potentiates NMJs for rapid activity-dependent structural plasticity that is achieved by muscle release of retrograde signals that regulate local presynaptic actin cytoskeletal dynamics (Piccioli, 2014).

Activity-dependent changes in synaptic structure play an important role in developmental wiring of the nervous system. The Drosophila larval neuromuscular junction (NMJ) has emerged as a model glutamatergic synapse that is well suited to study activity-dependent structural plasticity. The NMJ can be imaged in vivo during developmental periods of rapid synaptic growth when the axonal terminal expands ~5- to 10-fold in size over 5 d. Forward genetic screens to identify mutations that alter synaptic growth have revealed essential roles for retrograde bone morphogenic protein (BMP) signaling mediated by the secreted ligand Glass bottom boat (Gbb). Mutations that disrupt BMP signaling lead to synaptic undergrowth and neurotransmitter release defects. Multiple pathways downstream of retrograde BMP signaling through the type II receptor Wishful thinking (Wit) have been linked to synaptic growth, synapse stability, and homeostatic plasticity in Drosophila. BMP signaling via the Smad transcription factor Mothers against Dpp (Mad) regulates the expression of the Rho-type guanine nucleotide exchange factor (GEF) trio to control normal synaptic growth. Wit also interacts with LIM domain kinase 1 (Limk) to enhance synaptic stabilization in a pathway parallel to canonical Smad-dependent signaling. BMP signaling through Wit also potentiates synapses for homeostatic plasticity in a pathway that is independent of limk and synaptic growth regulation (Piccioli, 2014).

The NMJ displays acute structural plasticity in the form of rapid presynaptic bouton budding in response to elevated levels of neuronal activity. These rapidly generated presynaptic varicosities, referred to as ghost boutons, lack presynaptic and postsynaptic transmission machinery when initially formed. The budding of ghost boutons requires retrograde signaling mediated by the postsynaptic Ca2+-sensitive vesicle trafficking regulator synaptotagmin (Syt) 4 (Korkut, 2013). Syt4 also participates in developmental synaptic growth and controls retrograde signaling that mediates enhanced spontaneous release at the NMJ (Yoshihara, 2005; Barber, 2009). Beyond the role of Syt4 in ghost bouton budding, little is known about the signaling pathways that underlie this rapid form of structural synaptic plasticity. In particular, it is unclear whether pathways that regulate synaptic growth over the longer time scales of larval development also trigger acute structural plasticity. To address these issues, this study identified synaptic pathways that are required for rapid structural plasticity at Drosophila NMJs. Ghost bouton budding was found to be locally regulated at the synapse level, occurring in axons that have been severed from the neuronal cell body. In addition, activity-induced ghost bouton formation requires Syt1-mediated neurotransmitter release and postsynaptic glutamate receptor function. Like developmental growth, retrograde BMP signaling is required for ghost bouton budding. BMP signaling functions through a permissive role mediated by developmental Smad and Trio signaling, as well as through a local Wit-dependent modulation of Limk and Cofilin (Twinstar) activity that alters presynaptic actin dynamics (Piccioli, 2014).

Experimental analysis of ghost bouton budding at the Drosophila NMJ indicates that rapid activity-dependent synaptic growth requires retrograde BMP signaling at this synapse. The current data support a model in which BMP signaling through the type II receptor Wit is required developmentally to potentiate synapses for budding in response to elevated synaptic activity. This pathway requires Smad-dependent expression of the Rho-type GEF trio, and parallels a requirement for BMP signaling and Trio in developmental synaptic growth that occurs during the larval stages. In a parallel pathway, Wit interaction with Limk inhibits bouton budding through regulation of Cofilin activity. Both pathways regulate the synaptic actin cytoskeleton and may converge on similar actin regulatory molecules such as Limk and Cofilin via Rac1 or RhoA. Manipulating Cofilin activity levels by the overexpression of Limk or the expression of constitutively active/inactive Cofilin demonstrates that high Cofilin activity favors bouton budding, while low Cofilin activity inhibits budding. Local changes in the actin cytoskeleton that accompany activity-dependent bouton budding were also observed at sites of new synaptic growth. In addition, pharmacological disruption of normal actin turnover inhibits budding, suggesting that increased actin turnover mediated by Cofilin potentiates rapid activity-dependent synaptic plasticity (Piccioli, 2014).

Multiple genetic perturbations of BMP signaling were identified that altered the frequency of activity-dependent bouton budding at the NMJ. Although several of these mechanisms are shared with those previously characterized to control BMP-mediated developmental synaptic growth, several manipulations separated rapid activity-dependent BMP-mediated bouton budding from the slower forms of developmental growth. In the case of wit mutants or motor neuron overexpression of dad, a reduction in baseline bouton number was observed that showed varying degrees of severity. Wit mutants displayed strongly undergrown synapses, while dad overexpression animals had only modest synaptic undergrowth. In contrast, both these manipulations strongly suppressed ghost bouton budding. Additionally, synaptic undergrowth with partial knockdown of Gbb using postsynaptic RNAi was not observed, while this manipulation caused a strong reduction in ghost bouton budding. These observations indicate that rapid ghost bouton budding is more sensitive to modest perturbations in BMP signaling compared with developmental synaptic growth. One explanation for this differential sensitivity is that BMP signaling potentiates NMJs for activity-dependent bouton budding via transcriptional regulation of molecular components that are not required for normal synaptic growth. Alternatively, similar molecular pathways are required, but at different levels of output. In particular, trio mutants display a less severe synaptic undergrowth phenotype than wit mutants, but show similarly severe defects in ghost bouton budding. Because trio expression is strongly dependent on BMP signaling (Ball, 2010), a modest reduction in BMP output could reduce Trio levels such that ghost bouton budding is significantly reduced, while normal synaptic growth is less affected. It will be interesting to determine in future studies whether the developmental role for BMP signaling for acute structural plasticity shares a critical period as has recently been found for BMP function during developmental synaptic growth (Piccioli, 2014).

Given the requirement of the postsynaptic Ca2+ sensor Syt4 for normal levels of ghost bouton budding, an attractive model is that BMP is released acutely in response to elevated activity through the fusion of Syt4-positive postsynaptic vesicles. However, the current analysis indicates that retrograde BMP signaling through trio transcriptional upregulation is unlikely to be an instructive cue for bouton budding, as the severing of axons and the inhibition of retrograde trafficking of P-Mad before stimulation does not reduce budding in response to elevated activity. It is possible that synaptic P-Mad may play an instructional role in ghost bouton budding, as a local decrease in budding frequency was observed when Gbb expression was specifically reduced in muscle 6. Neuronal overexpression of dad also reduced synaptic P-Mad. Therefore, dad overexpression could inhibit ghost bouton budding by decreasing synaptic P-Mad signaling, in addition to decreasing nuclear Smad signaling. However, no dosage-dependent genetic interactions were observed between syt4 and wit, suggesting that Syt4 may participate in a separate pathway to regulate ghost bouton budding. Activity-dependent fusion of Syt4 postsynaptic vesicles (Yoshihara, 2005) could release a separate unidentified retrograde signal that provides an instructive cue for budding that would function in parallel to a developmental requirement for retrograde BMP signaling (Piccioli, 2014).

In addition to instructive cues from the postsynaptic compartment that trigger ghost bouton budding, the presynaptic nerve terminal must have molecular machines in place to read out these signals and execute the budding event. The regulation of Rho GTPases via Rho GEFs and GAPs downstream of extracellular cues is an attractive mechanism, as these proteins play critical roles in the regulation of neuronal morphology and axonal guidance. Several studies have shown that retrograde synaptic signaling regulates Rho GTPase activity to alter synaptic function and growth in Drosophila (Tolias, 2011). Ghost bouton budding mediated by developmental BMP signaling also shares some similarities with mechanisms underlying homeostatic plasticity at Drosophila NMJs. The Eph receptor is required for synaptic homeostasis at the NMJ, and it interfaces with developmental BMP signaling via Wit. While Eph receptor-mediated homeostatic plasticity predominantly requires the downstream RhoA-type GEF Ephexin, the Eph receptor may also signal through Rac1. Drosophila VAP-33A may also act as a ligand for synaptic Eph receptors, as it has been shown to regulate NMJ morphology and growth, while preferentially localizing to sites of bouton budding. The current analysis indicates that the levels of Trio, which functions as a Rho-type GEF, are bidirectionally correlated with ghost bouton budding activity and that overexpressed Trio is localized to ghost boutons after budding. As such, acute Trio regulation represents another attractive pathway for rapidly modifying bouton budding activity (Piccioli, 2014).

Rho GTPase signaling can produce distinct effects in differing systems and cell types depending on the presence or absence of downstream effectors, although most of these pathways ultimately impinge on regulation of the actin cytoskeletal. Indeed, this study has found a key role for Limk regulation of Cofilin activity in the control of ghost bouton budding. The current findings indicate that Limk activity normally functions to inhibit the formation of ghost boutons, as neuronal overexpression of Limk strongly suppressed activity-dependent bouton budding. Consistent with an inhibitory role for Limk, Cofilin activity promotes budding, while the overexpression of an inactive Cofilin inhibited budding. The expression of mutant Cofilin transgenes resulted in visible changes to the presynaptic actin cytoskeleton at NMJs, indicating that these manipulations likely alter rapid budding events by changing local actin dynamics at sites of potential growth. Using live imaging of F-actin dynamics before and after bouton budding, the formation of new F-actin puncta was observed at sites of bouton budding. Elevated Cofilin activity is sufficient to increase ghost bouton budding frequency, and is predicted to increase actin turnover and the formation of F-actin structures. Pharmacological disruption of actin polymerization dynamics also disrupts rapid bouton addition in response to elevated activity (Piccioli, 2014).

These findings support a model whereby Wit has opposing signaling roles with respect to bouton budding. Providing a permissive role via Smad signaling and an inhibitory role via Limk activation may provide for a system in which increased potential for rapid synaptic expansion is directly coupled to enhanced synaptic stability. This coupling could set a threshold for ghost bouton budding downstream of synaptic activity. In the background of moderate or low synaptic activity, Limk prevents ghost bouton budding. When synaptic activity is elevated, additional signaling events promote new synaptic growth by either reducing or outcompeting Limk activity, with a concurrent activation of Cofilin. Decreased Limk activity downstream of extracellular cues has been shown to regulate cell morphology in other systems as well, providing an attractive mechanism for rapid activity-dependent regulation of synaptic structure at Drosophila NMJs (Piccioli, 2014).

F-actin dismantling through a redox-driven synergy between Mical and cofilin

Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. This study uncovered a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties (Grintsevich, 2016).

This study found that Mical and cofilin function as a pair, synergizing in a Redox-dependent post-translational manner to disassemble F-actin and to control different cellular behaviors. Specifically, cofilin is a well-established actin regulatory protein and a relatively weak severer of F-actin. In contrast, Mical family Redox enzymes have only recently emerged downstream of Semaphorin-Plexin repellents as actin disassembly factors acting via the direct post-translational oxidation of actin. Previous work has also revealed that Mical, whose C-terminus associates with the intracellular portion of the Semaphorin transmembrane receptor plexin, binds with its N-terminal NADPH-dependent Redox domain to F-actin and selectively oxidizes actin's methionine-44 and 47 residues. It is proposed that Mical oxidation-induced changes in filament structure and/or dynamics improve cofilin's binding to actin filaments. This study also found that Mical-oxidized actin copolymers have different properties than unoxidized actin filaments. It is also known that the severing of actin filaments by cofilin is related to the mechanical properties of F-actin. The results support the idea that Mical uses oxidation to weaken the inter-actin (inter-protomer) contacts within filaments and these alterations dramatically speed up cofilin's ability to break/dismantle filaments. These results, therefore, uncover a previously unknown pathway of cellular F-actin disassembly and also present an unusual type of biological synergistic interaction -- one involving two different types of proteins (Mical and cofilin) and the Redox-dependent post-translational modification of a third protein (polymerized actin) (Grintsevich, 2016).

The results also shed new light on the mechanisms of action of both Mical and cofilin. They support a model that Mical and cofilin have been evolutionarily selected to work in tandem to ensure that even a low level of Mical activity in the presence of cofilin would facilitate F-actin disassembly, and vice versa. Moreover, unlike F-actin disassembly by cofilin, which promotes actin turnover by recirculation of monomers for polymerization, Mical post-translationally modifies actin, decreasing its capacity for re-polymerization until the oxidation is reversed. Thus, the Redox-driven synergy between Mical and cofilin not only rapidly disassembles F-actin but also generates post-translationally modified actin that re-assembles abnormally with a net effect of promoting F-actin instability. These results, therefore, provide important insights into how actin-based structures are rapidly and specifically dismantled in cells. Given their widespread overlapping expression patterns and diverse effects on cellular behaviors, this synergistic interaction between Mical and cofilin provides the molecular framework to rapidly dismantle multiple actin-based cellular structures (Grintsevich, 2016).

The branching code: A model of actin-driven dendrite arborization

The cytoskeleton is crucial for defining neuronal-type-specific dendrite morphologies. To explore how the complex interplay of actin-modulatory proteins (AMPs) can define neuronal types in vivo, this study focused on the class III dendritic arborization (c3da) neuron of Drosophila larvae. Using computational modeling, the main branches (MBs) of c3da neurons were demonstrated to follow general models based on optimal wiring principles, while the actin-enriched short terminal branches (STBs) require an additional growth program. To clarify the cellular mechanisms that define this second step, this study concentrated on STBs for an in-depth quantitative description of dendrite morphology and dynamics. Applying these methods systematically to mutants of six known and novel AMPs (Arp2/3, Capu, Ena, Singed, and Twinstar), the complementary roles were revealed of these individual AMPs in defining STB properties. These data suggest that diverse dendrite arbors result from a combination of optimal-wiring-related growth and individualized growth programs that are neuron-type specific (Sturner, 2022).

Neurons develop their dendrites in tight relation to their connection and computation requirements. Thus, dendrite morphologies display sophisticated type-specific patterns. From the cell biological and developmental perspective, this raises the question of at which level different neuronal types might use shared mechanisms to assemble their dendrites. And, conversely, how are specialized structures achieved in different neuronal types? To start addressing these question computational and comparative cell biological approaches were combined. It was found that two distinct growth programs are required to achieve models that faithfully reproduce the dendrite organization of c3da neurons. The models single out the STBs that are also molecularly identifiable as unique structures, displaying specific localization of actin and Singed. By combining time-lapse in vivo imaging and genetic analyses, this study sheds light on the machinery that controls the dynamic formation of those branchlets (Sturner, 2022).

The complex interplay of AMPs generates highly adaptive actin networks. In fact, in contrast to earlier unifying models, it is now clear that even the same cell can make more than one type of filopodium-like structure. This study characterized the effect of the loss of six AMPs on the morphology and dynamics of one specific type of dendritic branchlet, the STB of c3da neurons. With this information, a molecular model for branchlet dynamics in vivo is delineated in the developing animal. Similar approaches to model the molecular regulation of actin in dendrite filopodia have been taken recently for cultured neurons. The advantage of the present approach is that it relies directly on the effect of the loss of individual AMPs in vivo, preserving the morphology, dynamics, and adhesive properties of the branchlets, and non-cell-autonomous signals remain present (Sturner, 2022).

The combination of FRAP experiments and the localization of Singed/Fascin on the extending STBs indicated that actin is organized in a tight bundle of mostly uniparallel fibers in the STBs. This organization is thus very different from that of dendritic filopodia of hippocampal neurons in culture. The actin filaments in the bundle appear to be particularly stable in the c3da-neuron STBs, as the actin turnover that this study revealed by FRAP analysis was 4 times slower than that reported in dendrite spines of hippocampal neurons in vitro and 20-fold slower than in a lamellipodium of melanoma cells in vitro. It is nonetheless in line with previous data on stable c3da-neuron STBs and with bundled actin filaments of stress fibers of human osteosarcoma cells. Treadmilling was observed, similar to that of filopodia at the leading edge, with a retrograde flow rate 30 times slower than in filopodia of hippocampal cells and comparable to rates observed for developing neurons in culture lacking the mammalian homologues of Twinstar and actin-depolymerization factor (ADF)/Cofilin. Slower actin kinetics could be related to the fact that neurons differentiating in the complex 3D context of a developing animal are being imaged. Recent quantification of actin treadmilling in a growth cone of hippocampal neurons in 3D culture, however, did not produce differences with 2D-culture models(Sturner, 2022).

The alterations of MB and STB morphology and dynamics caused by the loss of individual AMP functions reported in this study can now be combined with preceding molecular knowledge about these conserved factors to produce a hypothetical model of the actin regulation underlying STB dynamics. Dendrite structure and time-lapse imaging point to an essential role of Twinstar/Cofilin for the initiation of a branchlet, in agreement with previous literature. Drosophila Twinstar/Cofilin is a member of the ADF/Cofilin protein family, with the capacity of severing actin filaments but with poor actin-filament-depolymerizing activity. It is thus proposed that Twinstar/Cofilin localized at the base of c3da STBs can induce a local fragmentation of actin filaments that can then be used as substrate by the Arp2/3 complex. In fact, in c4da neurons, Arp2/3 localizes transiently at the site where the branchlets will be formed, and its presence strongly correlates with the initiation of branchlet formation. Previous and present time-lapse data point to the role of Arp2/3 in the early phases of branchlet formation. Thus, it is suggested that localized activity of Arp2/3 generates a first localized membrane protrusion (Sturner, 2022).

Given the transitory localization of Arp2/3, this study interrogated the role of additional actin nucleators in this context. From an RNAi-supported investigation, Capu was identified as potential modifier of c3da STBs. Capu displays complex interactions with the actin-nucleator Spire during oogenesis, involving cooperative and independent functions of these two molecules. An increase in Spire levels correlates with a smaller dendritic tree and inappropriate, F-actin-rich, and shorter dendrites in c4da neurons. In this study, though, the loss of Spire function did not yield a detectable phenotype in c4da neurons. In c3da neurons, it was found that Capu and Spire support the formation of new branchlets and display a strong genetic interaction in the control of the number and length of MBs and STBs and surface area. Thus it is suggested that they cooperatively take over the nucleation of linear actin filaments possibly producing the bundle of uniparallel actin filaments. Mutants for capu showed changes in the positioning of dendritic branches, not observed in spire mutants, which could mean that Capu localization defines the sites of Capu/Spire activity. However, Spire seems to promote branch dynamics, suggesting additional independent functions of Spire possibly not related to nucleation, given that Spire itself is a weak actin nucleator. While there is no clear indication in vivo for the molecular mechanisms supporting this function, an actin-severing activity of Spire was reported in vitro. The role of Spire on STB dynamics appears to be consistent with favoring actin destabilization or actin dynamics (Sturner, 2022).

Singed/Fascin bundles actin filaments specifically in the c3da neuron STBs and gives these branches their straight conformation. The localization of Singed/Fascin in the c3da STBs correlates with their elongation. While the complete loss of singed function suppressed dynamics, the mild reduction in protein levels analyzed in this study led to more frequent STB elongations and retractions. Further, the branchlets extended at the wrong angles and displayed a tortuous path. Singed/Fascin controls the interaction of actin-filament bundles with Twinstar/Cofilin and can enhance Ena binding to barbed ends. Thus, in addition to generating mechanically rigid bundles, it can modulate actin dynamics by regulating the interaction of multiple AMPs with actin. It is speculated that the retraction and disappearance of the STB could be due to Singed/Fascin dissociating from the actin filaments, possibly in combination with Spire and Twinstar/Cofilin additionally severing actin filaments. In fact, the presence of detectable Twinstar/Cofilin along the c3da STBs was recently reported (Sturner, 2022).

Ena is important for restricting STB length, and it inhibits the new formation and extension of STBs. This appears to be a surprising function for Ena that is in contrast to its role in promoting actin-filament elongation or to its capacity of supporting the activation of the WAVE regulatory complex. Similar to what was previously reported for ena-mutant c4da neurons, a balance between elongation and branching was also observed in c3da neurons. In Drosophila macrophages, Ena was shown to associate with Singed/Fascin within lamellipodia. In line with these recent data, it is suggested that Ena might closely cooperate with Singed to form tight actin bundles that slow down STB elongation (Sturner, 2022).

Taken together, a comprehensive molecular model of dendrite-branch dynamics for the STBs of c3da neurons was put forward. In this analysis, the role of extracellular signals on the regulation of the dynamics of STBs was excluded, for simplicity. Nonetheless, such signals are likely to have a profound effect, particularly on the regulation of elongation and stabilization of STBs in relation to their target substrate. In addition, similar to what has been suggested for c1da neurons, the distribution of MBs in the target area might follow guidance cues that were not included in the analysis, such as permissive signals that specifically guide c4da neurons to tile the body wall or promote appropriate space filling (Sturner, 2022).

The investigation of morphological parameters in combination with genetic analysis has proven extremely powerful to reveal initial molecular mechanisms of dendrite differentiation. Early studies, though, have been limited in the description power of their analysis concentrating on just one or two parameters (e.g., number of termini and total dendrite length). This limitation has been recognized and addressed in more recent studies (Sturner, 2022).

A major outcome of the present and previous work is the establishment of powerful tools for a thorough and comparative quantitative morphological analysis of different mutant groups. A detailed tracing of neuronal dendrites of the entire dendritic tree or a certain area of the tree in a time series with a subsequent automatic analysis allows a precise description of mutant phenotypes. This study additionally generated tools for extracting quantitative parameters of the dynamic behavior of dendrite branches from time-lapse movies based on a novel branch registration software. This time-lapse tool yields an automated quantification after registration detecting branch types and their dynamics. Moreover, the tool operates in the same framework as the tracing and morphological analysis. These tools available within the TREES toolbox, and their use to support comparative analysis among datasets is encouraged (Sturner, 2022).

What are the fundamental principles that define dendrite elaboration and which constraints need to be respected by neurons in establishing their complex arbors? Models based on local or global rules have been applied to reproduce the overall organization of dendritic trees, including da neurons. The c3da model is based on the fundamental organizing principle that dendrites are built through minimizing cable length and signal conduction times. This general rule for optimal wiring predicts tight scaling relationships between fundamental branching statistics, such as the number of branches, the total length, and the dendrite's spanning field (Sturner, 2022).

This study found that c3da neurons respect the general developmental SFGT or MST models when stripped of all their STBs. However, the characteristic STBs of c3da dendrites did not follow this scaling behavior. Instead, a second growth program had to be applied to add the STBs to this basic structure, respecting their number, total length, and distribution. The two-step model developed in this work suggests that while main dendritic trees have common growth rules, the dendritic specializations of different neuronal cell types do not necessarily have the same constraints. This view is compatible with findings in a companion paper showing, in c1da neurons, a specialized branch-retraction step following an initial growth step. In the two-step c3da dendrite model, the resulting synthetic morphologies resemble the real dendritic trees including those of five out of the six AMP mutant dendritic trees without any changes to the model parameters. The two-step model uses, for example, the reduced total length and reduced surface area of mutants for singed and twinstar and grows synthetic trees that have the same distribution of branch lengths and amounts as expected for those mutants. The synthetic trees corresponding to the twinstar mutant have less STBs than any other AMP mutant synthetic tree, consistent with the real mutant phenotypes (Sturner, 2022).

This work indicates that a combination of thorough statistical analysis (such as using the presented morphometrics) and models, like the one developed in this study, can help capture the fundamental principles that govern dendrite differentiation. Together with genetics analysis and systematic cell biology approaches, this type of study can deliver quantitative predictions for molecular models of dendrite elaboration (Sturner, 2022).

In conclusion, this study has put forward the hypothesis that neuronal dendrites are built based on common, shared growth programs. An additional refinement step is then added to this scaffold, allowing each neuron type to specialize based on its distinctive needs in terms of number and distribution of inputs. In the exemplary case of c3da neurons, this study investigated molecular properties of these more-specialized growth programs and proposed a first comprehensive model of actin regulation that explains the morphology and dynamics of branchlets (Sturner, 2022).

Most of the AMPs studied are essential, and all perform multiple functions during the course of development. Clearly, in these experiments, the acute function of each AMP in the process of STB formation and during STB dynamics has not been isolated. Rather, the progressive reduction of functional protein in MARCM clones or during the development of homozygous animals might represent a confounding factor. Future studies will be aimed at using and developing tools for acute protein-function inactivation in vivo to add to the toolbox (Sturner, 2022).

GENE STRUCTURE

cDNA clone length - 771

Bases in 5' UTR - 168

Exons - 4

Bases in 3' UTR - 156

PROTEIN STRUCTURE

Amino Acids - 148

Structural Domains

See SMART (Simple Modular Architecture Research Tool) for information of Twinstar structure.

date revised: 25 April 2024

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