Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αß neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αß neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

Nucleation causes an actin network to fragment into multiple high-density domains.

Actin networks rely on nucleation mechanisms to generate new filaments because spontaneous nucleation is kinetically disfavored. Branching nucleation of actin filaments by actin-related protein (Arp2/3), in particular, is critical for actin self-organization. In this study, we use the simulation platform for active matter MEDYAN to generate 2000 s long stochastic trajectories of actin networks, under varying Arp2/3 concentrations, in reaction volumes of biologically meaningful size (>20 µm3). We find that the dynamics of Arp2/3 increase the abundance of short filaments and increases network treadmilling rate. By analyzing the density fields of F-actin, we find that at low Arp2/3 concentrations, F-actin is organized into a single connected and contractile domain, while at elevated Arp2/3 levels (10 nM and above), such high-density actin domains fragment into smaller domains spanning a wide range of volumes. These fragmented domains are extremely dynamic, continuously merging and splitting, owing to the high treadmilling rate of the underlying actin network. Treating the domain dynamics as a drift-diffusion process, we find that the fragmented state is stochastically favored, and the network state slowly drifts toward the fragmented state with considerable diffusion (variability) in the number of domains. We suggest that tuning the Arp2/3 concentration enables cells to transition from a globally coherent cytoskeleton, whose response involves the entire cytoplasmic network, to a fragmented cytoskeleton, where domains can respond independently to locally varying signals.

Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.

The Abl pathway bifurcates to balance Enabled and Rac signaling in axon patterning in Drosophila.

The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. Although individual components are known, the relationships among them remain unresolved. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin-regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain. Finally, we show that a key target of Abl signaling in axons is the WAVE complex that promotes the formation of branched actin networks. Thus, we show that Abl constitutes a bifurcating network, suppressing Ena activity in parallel with stimulation of WAVE. We suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning.

Phosphorylation and Proteolytic Cleavage of Notch in Canonical and Noncanonical Notch Signaling.

The Notch signaling pathway seems deceptively simple, with its key feature being a direct connection between extracellular signal and transcriptional output without the need for an extended chain of protein intermediaries as required by so many other signaling paradigms. However, this apparent simplicity hides considerable complexity. Consistent with its central role in many aspects of development, Notch signaling has an extensive collection of mechanisms that it employs alongside of its core transcriptional machinery. These so-called noncanonical Notch pathways diversify the potential outputs of Notch, and allow it to coordinate regulation of many aspects of the biology of cells. Here we will review noncanonical Notch signaling with special attention to the role of posttranslational modifications of Notch. We will also consider the importance of coordinating the activity of gene expression with regulation of cell morphology in biological processes, including axon guidance and other morphological events during embryogenesis.

A role for actomyosin contractility in Notch signaling.

BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.

Dendrite architecture organized by transcriptional control of the F-actin nucleator Spire.

The architectures of dendritic trees are crucial for the wiring and function of neuronal circuits because they determine coverage of receptive territories, as well as the nature and strength of sensory or synaptic inputs. Here, we describe a cell-intrinsic pathway sculpting dendritic arborization (da) neurons in Drosophila that requires Longitudinals Lacking (Lola), a BTB/POZ transcription factor, and its control of the F-actin cytoskeleton through Spire (Spir), an actin nucleation protein. Loss of Lola from da neurons reduced the overall length of dendritic arbors, increased the expression of Spir, and produced inappropriate F-actin-rich dendrites at positions too near the cell soma. Selective removal of Lola from only class IV da neurons decreased the evasive responses of larvae to nociception. The increased Spir expression contributed to the abnormal F-actin-rich dendrites and the decreased nocifensive responses because both were suppressed by reduced dose of Spir. Thus, an important role of Lola is to limit expression of Spir to appropriate levels within da neurons. We found Spir to be expressed in dendritic arbors and to be important for their development. Removal of Spir from class IV da neurons reduced F-actin levels and total branch number, shifted the position of greatest branch density away from the cell soma, and compromised nocifensive behavior. We conclude that the Lola-Spir pathway is crucial for the spatial arrangement of branches within dendritic trees and for neural circuit function because it provides balanced control of the F-actin cytoskeleton.

Cdk5 regulates developmental remodeling of mushroom body neurons in Drosophila.

BACKGROUND: During metamorphosis, axons and dendrites of the mushroom body (MB) in the Drosophila central brain are remodeled extensively to support the transition from larval to adult behaviors. RESULTS: We show here that the neuronal cyclin-dependent kinase, Cdk5, regulates the timing and rate of mushroom body remodeling: reduced Cdk5 activity causes a delay in pruning of MB neurites, while hyperactivation accelerates it. We further show that Cdk5 cooperates with the ubiquitin-proteasome system in this process. Finally, we show that Cdk5 modulates the first overt step in neurite disassembly, dissolution of the neuronal tubulin cytoskeleton, and provide evidence that it also acts at additional steps of MB pruning. CONCLUSIONS: These data show that Cdk5 regulates the onset and extent of remodeling of the Drosophila MB. Given the wide phylogenetic conservation of Cdk5, we suggest that it is likely to play a role in developmental remodeling in other systems, as well. Moreover, we speculate that the well-established role of Cdk5 in neurodegeneration may involve some of the same cellular mechanisms that it uses during developmental remodeling.

How Notch establishes longitudinal axon connections between successive segments of the Drosophila CNS.

Development of the segmented central nerve cords of vertebrates and invertebrates requires connecting successive neuromeres. Here, we show both how a pathway is constructed to guide pioneer axons between segments of the Drosophila CNS, and how motility of the pioneers along that pathway is promoted. First, canonical Notch signaling in specialized glial cells causes nearby differentiating neurons to extrude a mesh of fine projections, and shapes that mesh into a continuous carpet that bridges from segment to segment, hugging the glial surface. This is the direct substratum that pioneer axons follow as they grow. Simultaneously, Notch uses an alternate, non-canonical signaling pathway in the pioneer growth cones themselves, promoting their motility by suppressing Abl signaling to stimulate filopodial growth while presumably reducing substratum adhesion. This propels the axons as they establish the connection between successive segments.

MANIPULATING MITOCHONDRIAL REACTIVE OXYGEN SPECIES ALTERS SURVIVAL IN UNEXPECTED WAYS IN A DROSOPHILA MODEL OF NEURODEGENERATION.

Reactive oxygen species (ROS) are associated with aging and neurodegeneration, but the significance of this association remains obscure. Here, using a Drosophila model of age-related neurodegeneration, we probe this relationship in the pathologically relevant tissue, the brain, by quantifying three specific mitochondrial ROS and manipulating these redox species pharmacologically. Our goal is to ask whether pathology-associated changes in redox state are detrimental for survival, whether they may be beneficial responses, or whether they are simply covariates of pathology that do not alter viability. We find, surprisingly, that increasing mitochondrial H2O2 correlates with improved survival. We also find evidence that drugs that alter the mitochondrial glutathione redox potential modulate survival primarily through the compensatory effects they induce rather than through their direct effects on the final mitochondrial glutathione redox potential per se. We also find that the response to treatment with a redox-altering drug varies dramatically depending on the age at which the drug is administered, the duration of the treatment, and the genotype of the individual receiving the drug. These data have important implications for the design and interpretation of studies investigating the effect of redox state on health and disease as well as on efforts to modify the redox state to achieve therapeutic goals.

Manipulating mitochondrial reactive oxygen species alters survival in unexpected ways in a Drosophila Cdk5 model of neurodegeneration.

Reactive oxygen species (ROS) are associated with aging and neurodegeneration, but the significance of this association remains obscure. Here, using a Drosophila Cdk5 model of age-related neurodegeneration, we probe this relationship in the pathologically relevant tissue, the brain, by quantifying three specific mitochondrial ROS and manipulating these redox species pharmacologically. Our goal is to ask whether pathology-associated changes in redox state are detrimental for survival, whether they may be beneficial responses to pathology, or whether they are covariates of pathology that do not alter viability. We find, surprisingly, that increasing mitochondrial H2O2 correlates with improved survival. We also find evidence that drugs that alter the mitochondrial glutathione redox potential modulate survival primarily through the compensatory effects they induce rather than through their direct effects on the final mitochondrial glutathione redox potential. We also find that the response to treatment with a redox-altering drug varies depending on the age and genotype of the individual receiving the drug as well as the duration of the treatment. These data have important implications for the design and interpretation of studies investigating the effect of redox state on health and disease as well as on efforts to modify the redox state to achieve therapeutic goals.

Changes in mitochondrial distribution occur at the axon initial segment in association with neurodegeneration in Drosophila.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic vs. axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

Changes in mitochondrial distribution occur at the axon initial segment in association with neurodegeneration in Drosophila.

Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, we investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. We find that reducing Cdk5 activity does not alter the balance of mitochondrial localization to the somatodendritic versus axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. We do, however, observe changes in mitochondrial distribution at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, we observe that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

Disabled is a bona fide component of the Abl signaling network.

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.

A new view of axon growth and guidance grounded in the stochastic dynamics of actin networks.

The mechanism of axon growth and guidance is a core, unsolved problem in neuroscience and cell biology. For nearly three decades, our view of this process has largely been based on deterministic models of motility derived from studies of neurons cultured in vitro on rigid substrates. Here, we suggest a fundamentally different, inherently probabilistic model of axon growth, one that is grounded in the stochastic dynamics of actin networks. This perspective is motivated and supported by a synthesis of results from live imaging of a specific axon growing in its native tissue in vivo, together with single-molecule computational simulations of actin dynamics. In particular, we show how axon growth arises from a small spatial bias in the intrinsic fluctuations of the axonal actin cytoskeleton, one that produces net translocation of the axonal actin network by differentially modulating local probabilities of network expansion versus compaction. We discuss the relationship between this model and current views of axon growth and guidance mechanism and demonstrate how it offers explanations for various longstanding puzzles in this field. We further point out the implications of the probabilistic nature of actin dynamics for many other processes of cell morphology and motility.

What do we mean by "aging"? Questions and perspectives revealed by studies in Drosophila.

What is the nature of aging, and how best can we study it? Here, using a series of questions that highlight differing perspectives about the nature of aging, we ask how data from Drosophila melanogaster at the organismal, tissue, cellular, and molecular levels shed light on the complex interactions among the phenotypes associated with aging. Should aging be viewed as an individual's increasing probability of mortality over time or as a progression of physiological states? Are all age-correlated changes in physiology detrimental to vigor or are some compensatory changes that maintain vigor? Why do different age-correlated functions seem to change at different rates in a single individual as it ages? Should aging be considered as a single, integrated process across the scales of biological resolution, from organismal to molecular, or must we consider each level of biological scale as a separate, distinct entity? Viewing aging from these differing perspectives yields distinct but complementary interpretations about the properties and mechanisms of aging and may offer a path through the complexities related to understanding the nature of aging.

Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Cdk5 regulates the size of an axon initial segment-like compartment in mushroom body neurons of the Drosophila central brain.

The axon initial segment (AIS) is the specialized compartment of vertebrate axons where action potentials are initiated. Despite longtime attention to the unique functions of this compartment, the mechanisms that regulate AIS formation and maintenance are not known. Here, we identify a novel compartment in Drosophila mushroom body neurons that mirrors the molecular hallmarks of the vertebrate AIS as judged by accumulation of the anchoring protein Ankyrin1, presence of a specialized actin cytoskeleton, exclusion of both axon-specific and somatodendritic-specific cell surface proteins, and accumulation of a unique combination of voltage-gated ion channels. Using pharmacological treatments, we show that, similar to the vertebrate AIS, the integrity of this region of γ-neurons and its ability to tether membrane proteins depends on an intact actin cytoskeleton. We further show that Cdk5/p35 kinase regulates the formation and maintenance of the putative AIS by controlling the position of its distal boundary. Thus, boosting Cdk5 activity in γ-neurons extends the AIS by as much as 100%, while eliminating Cdk5 activity causes the domain to shrink proximally or disappear altogether. These data demonstrate that Cdk5/p35 kinase is a key regulator of the development and maintenance of the AIS in Drosophila.

Noncanonical Notch function in motor axon guidance is mediated by Rac GTPase and the GEF1 domain of Trio.

The receptor Notch interacts with the Abl tyrosine kinase signaling pathway to control axon growth and guidance in Drosophila motor neurons. In part, this is mediated by binding to Trio, a guanine nucleotide exchange factor (GEF) for Rho GTPases. We show here that one of the two GEF domains of Trio, the Rac-specific GEF1, is essential for Trio-dependent motor axon guidance and for the genetic suppression of Notch function in motor axon patterning, but the Rho-specific GEF2 domain is not. Consistent with this, we show that Rac, and not Rho1 or Cdc42, interacts genetically with Notch in a manner indistinguishable from that of bona fide Abl signaling components. We infer, therefore, that Rac is a key component of Abl signaling in Drosophila motor axons, and specifically that it is the crucial Rho GTPase in "noncanonical" Notch/Abl signaling.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.