The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons.

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.

Science communication in the field of fundamental biomedical research (editorial).

The aim of this special issue on science communication is to inspire and help scientists who are taking part or want to take part in science communication and engage with the wider public, clinicians, other scientists or policy makers. For this, some articles provide concise and accessible advice to individual scientists, science networks, or learned societies on how to communicate effectively; others share rationales, objectives and aims, experiences, implementation strategies and resources derived from existing long-term science communication initiatives. Although this issue is primarily addressing scientists working in the field of biomedical research, much of it similarly applies to scientists from other disciplines. Furthermore, we hope that this issue will also be used as a helpful resource by academic science communicators and social scientists, as a collection that highlights some of the major communication challenges that the biomedical sciences face, and which provides interesting case studies of initiatives that use a breadth of strategies to address these challenges. In this editorial, we first discuss why we should communicate our science and contemplate some of the different approaches, aspirations and definitions of science communication. We then address the specific challenges that researchers in the biomedical sciences are faced with when engaging with wider audiences. Finally, we explain the rationales and contents of the different articles in this issue and the various science communication initiatives and strategies discussed in each of them, whilst also providing some information on the wide range of further science communication activities in the biomedical sciences that could not all be covered here.

The Manchester Fly Facility: Implementing an objective-driven long-term science communication initiative.

Science communication is increasingly important for scientists, although research, teaching and administration activities tend to eat up our time already, and budgets for science communication are usually low. It appears impossible to combine all these tasks and, in addition, to develop engagement activities to a quality and impact that would make the efforts worth their while. Here we argue that these challenges are easier addressed when centering science communication initiatives on a long-term vision with a view to eventually forming outreach networks where the load can be shared whilst being driven to higher momentum. As one example, we explain the science communication initiative of the Manchester Fly Facility. It aims to promote public awareness of research using the model organism Drosophila, which is a timely, economic and most efficient experimental strategy to drive discovery processes in the biomedical sciences and must have a firm place in the portfolios of funding organisations. Although this initiative by the Manchester Fly Facility is sustained on a low budget, its long-term vision has allowed gradual development into a multifaceted initiative: (1) targeting university students via resources and strategies for the advanced training in fly genetics; (2) targeting the general public via science fairs, educational YouTube videos, school visits, teacher seminars and the droso4schools project; (3) disseminating and marketing strategies and resources to the public as well as fellow scientists via dedicated websites, blogs, journal articles, conference presentations and workshops - with a view to gradually forming networks of drosophilists that will have a greater potential to drive the science communication objective to momentum and impact. Here we explain the rationales and implementation strategies for our various science communication activities - which are similarly applicable to other model animals and other areas of academic science - and share our experiences and resources to provide ideas and readily available means to those who are actively engaging or intend to do so.

The droso4schools project: Long-term scientist-teacher collaborations to promote science communication and education in schools.

Science communication is becoming an increasingly important part of a scientist's remit, and engaging with primary and secondary schools is one frequently chosen strategy. Here we argue that science communication in schools will be more effective if based on good understanding of the realities of school life, which can be achieved through structured participation and/or collaboration with teachers. For example, the Manchester Fly Facility advocates the use of the fruit fly Drosophila as an important research strategy for the discovery processes in the biomedical sciences. To communicate this concept also in schools, we developed the 'droso4schools' project as a refined form of scientist-teacher collaboration that embraces the expertise and interests of teachers. Within this project, we place university students as teaching assistants in university partner schools to collaborate with teachers and develop biology lessons with adjunct support materials. These lessons teach curriculum-relevant biology topics by making use of the profound conceptual understanding existing in Drosophila combined with parallel examples taken from human biology. By performing easy to implement experiments with flies, we bring living organisms into these lessons, thus endeavouring to further enhance the pupil's learning experience. In this way, we do not talk about flies but rather work with flies as powerful teaching tools to convey mainstream curriculum biology content, whilst also bringing across the relevance of Drosophila research. Through making these lessons freely available online, they have the potential to reach out to teachers and scientists worldwide. In this paper, we share our experiences and strategies to provide ideas for scientists engaging with schools, including the application of the droso4schools project as a paradigm for long-term school engagement which can be adapted also to other areas of science.

Drosophila Short stop as a paradigm for the role and regulation of spectraplakins.

Spectraplakins are evolutionarily well conserved cytoskeletal linker molecules that are true members of three protein families: plakins, spectrins and Gas2-like proteins. Spectraplakin genes encode at least 7 characteristic functional domains which are combined in a modular fashion into multiple isoforms, and which are responsible for an enormous breadth of cellular functions. These functions are related to the regulation of actin, microtubules, intermediate filaments, intracellular organelles, cell adhesions and signalling processes during the development and maintenance of a wide variety of tissues. To gain a deeper understanding of this enormous functional diversity, invertebrate genetic model organisms, such as the fruit fly Drosophila, can be used to develop concepts and mechanistic paradigms that can inform the investigation in higher animals or humans. Here we provide a comprehensive overview of our current knowledge of the Drosophila spectraplakin Short stop (Shot). We describe its functional domains and isoforms and compare them with those of the mammalian spectraplakins dystonin and MACF1. We then summarise its roles during the development and maintenance of the nervous system, epithelia, oocytes and muscles, taking care to compare and contrast mechanistic insights across these functions in the fly, but especially also with related functions of dystonin and MACF1 in mostly mammalian contexts. We hope that this review will improve the wider appreciation of how work on Drosophila Shot can be used as an efficient strategy to promote the fundamental concepts and mechanisms that underpin spectraplakin functions, with important implications for biomedical research into human disease.

Curvature-Sensitive Kinesin Binding Can Explain Microtubule Ring Formation and Reveals Chaotic Dynamics in a Mathematical Model.

Microtubules are filamentous tubular protein polymers which are essential for a range of cellular behaviour, and are generally straight over micron length scales. However, in some gliding assays, where microtubules move over a carpet of molecular motors, individual microtubules can also form tight arcs or rings, even in the absence of crosslinking proteins. Understanding this phenomenon may provide important explanations for similar highly curved microtubules which can be found in nerve cells undergoing neurodegeneration. We propose a model for gliding assays where the kinesins moving the microtubules over the surface induce ring formation through differential binding, substantiated by recent findings that a mutant version of the motor protein kinesin applied in solution is able to lock-in microtubule curvature. For certain parameter regimes, our model predicts that both straight and curved microtubules can exist simultaneously as stable steady states, as has been seen experimentally. Additionally, unsteady solutions are found, where a wave of differential binding propagates down the microtubule as it glides across the surface, which can lead to chaotic motion. Whilst this model explains two-dimensional microtubule behaviour in an experimental gliding assay, it has the potential to be adapted to explain pathological curling in nerve cells.

Using fly genetics to dissect the cytoskeletal machinery of neurons during axonal growth and maintenance.

The extension of long slender axons is a key process of neuronal circuit formation, both during brain development and regeneration. For this, growth cones at the tips of axons are guided towards their correct target cells by signals. Growth cone behaviour downstream of these signals is implemented by their actin and microtubule cytoskeleton. In the first part of this Commentary, we discuss the fundamental roles of the cytoskeleton during axon growth. We present the various classes of actin- and microtubule-binding proteins that regulate the cytoskeleton, and highlight the important gaps in our understanding of how these proteins functionally integrate into the complex machinery that implements growth cone behaviour. Deciphering such machinery requires multidisciplinary approaches, including genetics and the use of simple model organisms. In the second part of this Commentary, we discuss how the application of combinatorial genetics in the versatile genetic model organism Drosophila melanogaster has started to contribute to the understanding of actin and microtubule regulation during axon growth. Using the example of dystonin-linked neuron degeneration, we explain how knowledge acquired by studying axonal growth in flies can also deliver new understanding in other aspects of neuron biology, such as axon maintenance in higher animals and humans.

The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline.

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.

Drosophila Psidin regulates olfactory neuron number and axon targeting through two distinct molecular mechanisms.

The formation of neuronal circuits is a key process of development, laying foundations for behavior. The cellular mechanisms regulating circuit development are not fully understood. Here, we reveal Psidin as an intracellular regulator of Drosophila olfactory system formation. We show that Psidin is required in several classes of olfactory receptor neurons (ORNs) for survival and subsequently for axon guidance. During axon guidance, Psidin functions as an actin regulator and antagonist of Tropomyosin. Accordingly, Psidin-deficient primary neurons in culture display growth cones with significantly smaller lamellipodia. This lamellipodial phenotype, as well as the mistargeting defects in vivo, is suppressed by parallel removal of Tropomyosin. In contrast, Psidin functions as the noncatalytic subunit of the N-acetyltransferase complex B (NatB) to maintain the number of ORNs. Psidin physically binds the catalytic NatB subunit CG14222 (dNAA20) and functionally interacts with it in vivo. We define the dNAA20 interaction domain within Psidin and identify a conserved serine as a candidate for phosphorylation-mediated regulation of NatB complex formation. A phosphomimetic mutation of this serine showed severely reduced binding to dNAA20 in vitro. In vivo, it fully rescued the targeting defect but not the reduction in neuron numbers. In addition, we show that a different amino acid point mutation shows exactly the opposite effect by rescuing only the cell number but not the axon targeting defect. Together, our data suggest that Psidin plays two independent developmental roles via the acquisition of separate signaling pathways, both of which contribute to the formation of olfactory circuits.

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

Maintaining essential microtubule bundles in meter-long axons: a role for local tubulin biogenesis?

Axons are the narrow, up-to-meter long cellular processes of neurons that form the biological cables wiring our nervous system. Most axons must survive for an organism's lifetime, i.e. up to a century in humans. Axonal maintenance depends on loose bundles of microtubules that run without interruption all along axons. The continued turn-over and the extension of microtubule bundles during developmental, regenerative or plastic growth requires the availability of α/ß-tubulin heterodimers up to a meter away from the cell body. The underlying regulation in axons is poorly understood and hardly features in past and contemporary research. Here we discuss potential mechanisms, particularly focussing on the possibility of local tubulin biogenesis in axons. Current knowledge might suggest that local translation of tubulin takes place in axons, but far less is known about the post-translational machinery of tubulin biogenesis involving three chaperone complexes: prefoldin, CCT and TBC. We discuss functional understanding of these chaperones from a range of model organisms including yeast, plants, flies and mice, and explain what is known from human diseases. Microtubules across species depend on these chaperones, and they are clearly required in the nervous system. However, most chaperones display a high degree of functional pleiotropy, partly through independent functions of individual subunits outside their complexes, thus posing a challenge to experimental studies. Notably, we found hardly any studies that investigate their presence and function particularly in axons, thus highlighting an important gap in our understanding of axon biology and pathology.

How neurons maintain their axons long-term: an integrated view of axon biology and pathology.

Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery-all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the 'dependency cycle of local axon homeostasis' as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases.

The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

Microtubule regulation: Transcending the tenet of K40 acetylation.

For 36 years, the acetylation of lysine 40 in α-tubulin has provided the paradigm for how post-translational acetylation stabilises microtubules. A new study demonstrates that acetylation of lysine 394 in α-tubulin also mediates microtubule stabilisation in neurons.

Re-evaluating the actin-dependence of spectraplakin functions during axon growth and maintenance.

Axons are the long and slender processes of neurons constituting the biological cables that wire the nervous system. The growth and maintenance of axons require loose microtubule bundles that extend through their entire length. Understanding microtubule regulation is therefore an essential aspect of axon biology. Key regulators of neuronal microtubules are the spectraplakins, a well-conserved family of cytoskeletal cross-linkers that underlie neuropathies in mouse and humans. Spectraplakin deficiency in mouse or Drosophila causes severe decay of microtubule bundles and reduced axon growth. The underlying mechanisms are best understood for Drosophila's spectraplakin Short stop (Shot) and believed to involve cytoskeletal cross-linkage: Shot's binding to microtubules and Eb1 via its C-terminus has been thoroughly investigated, whereas its F-actin interaction via N-terminal calponin homology (CH) domains is little understood. Here, we have gained new understanding by showing that the F-actin interaction must be finely balanced: altering the properties of F-actin networks or deleting/exchanging Shot's CH domains induces changes in Shot function-with a Lifeact-containing Shot variant causing remarkable remodeling of neuronal microtubules. In addition to actin-microtubule (MT) cross-linkage, we find strong indications that Shot executes redundant MT bundle-promoting roles that are F-actin-independent. We argue that these likely involve the neuronal Shot-PH isoform, which is characterized by a large, unexplored central plakin repeat region (PRR) similarly existing also in mammalian spectraplakins.

A common theme for axonopathies? The dependency cycle of local axon homeostasis.

The number of acquired or inherited conditions leading to axon degeneration (from now on referred to as axonopathies) is vast. To diagnose patients, clinicians use a range of indicators including physiology, morphology, family and patient history, as well as genetics, with the specific location of the lesion within the nervous system being a prominent feature. For the neurobiologist, these criteria are often unsatisfactory, and key questions remain unanswered. For example, does it make sense that different axonopathies affect distinct neuron groups through distinct mechanisms? Would it not be more likely that there are common routes to axon degeneration? In this opinion piece, I shall pose this fundamental question and try to find answers that are hopefully thought-provoking and trigger some conceptual rethinking in the field. I will conclude by describing the 'dependency cycle of axon homeostasis' as a new approach to make sense of the intricate connections of axon biology and physiology, also suggesting that different axonopathies might share common paths to axon degeneration.

Cytoskeletal organization of axons in vertebrates and invertebrates.

The maintenance of axons for the lifetime of an organism requires an axonal cytoskeleton that is robust but also flexible to adapt to mechanical challenges and to support plastic changes of axon morphology. Furthermore, cytoskeletal organization has to adapt to axons of dramatically different dimensions, and to their compartment-specific requirements in the axon initial segment, in the axon shaft, at synapses or in growth cones. To understand how the cytoskeleton caters to these different demands, this review summarizes five decades of electron microscopic studies. It focuses on the organization of microtubules and neurofilaments in axon shafts in both vertebrate and invertebrate neurons, as well as the axon initial segments of vertebrate motor- and interneurons. Findings from these ultrastructural studies are being interpreted here on the basis of our contemporary molecular understanding. They strongly suggest that axon architecture in animals as diverse as arthropods and vertebrates is dependent on loosely cross-linked bundles of microtubules running all along axons, with only minor roles played by neurofilaments.

Formin proteins of the DAAM subfamily play a role during axon growth.

The regulation of growth cone actin dynamics is a critical aspect of axonal growth control. Among the proteins that are directly involved in the regulation of actin dynamics, actin nucleation factors play a pivotal role by promoting the formation of novel actin filaments. However, the essential nucleation factors in developing neurons have so far not been clearly identified. Here, we show expression data, and use true loss-of-function analysis and targeted expression of activated constructs to demonstrate that the Drosophila formin DAAM plays a critical role in axonal morphogenesis. In agreement with this finding, we show that dDAAM is required for filopodia formation at axonal growth cones. Our genetic interaction, immunoprecipitation and protein localization studies argue that dDAAM acts in concert with Rac GTPases, Profilin and Enabled during axonal growth regulation. We also show that mouse Daam1 rescues the CNS defects observed in dDAAM mutant flies to a high degree, and vice versa, that Drosophila DAAM induces the formation of neurite-like protrusions when expressed in mouse P19 cells, strongly suggesting that the function of DAAM in developing neurons has been conserved during evolution.