Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons.

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. We investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. We used CRISPR-Cas9 genome engineering approaches to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, we found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. Our data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on our data, we propose that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

A CRISPR view of development.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development.

Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function.

A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, we discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, our results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the "histone code," in regulating dendrite morphology.

Effects of mutating α-tubulin lysine 40 on sensory dendrite development.

Microtubules are essential for neuronal structure and function. Axonal and dendritic microtubules are enriched in post-translational modifications that impact microtubule dynamics, transport and microtubule-associated proteins. Acetylation of α-tubulin lysine 40 (K40) is a prominent and conserved modification of neuronal microtubules. However, the cellular role of microtubule acetylation remains controversial. To resolve how microtubule acetylation might affect neuronal morphogenesis, we mutated endogenous α-tubulin in vivo using a new Drosophila strain that facilitates the rapid knock-in of designer αTub84B alleles (the predominant α-tubulin-encoding gene in flies). Leveraging our new strain, we found that microtubule acetylation, as well as polyglutamylation and (de)tyrosination, is not essential for survival. However, we found that dendrite branch refinement in sensory neurons relies on α-tubulin K40. Mutagenesis of K40 reveals moderate yet significant changes in dendritic lysosome transport, microtubule polymerization and Futsch protein distribution in dendrites but not in axons. Our studies point to an unappreciated role for α-tubulin K40 and acetylation in dendrite morphogenesis. While our results are consistent with the idea that acetylation tunes microtubule function within neurons, they also suggest there may be an acetylation-independent requirement for α-tubulin K40.This article has an associated First Person interview with the first author of the paper.

Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

Role of kinesin-1-based microtubule sliding in Drosophila nervous system development.

The plus-end microtubule (MT) motor kinesin-1 is essential for normal development, with key roles in the nervous system. Kinesin-1 drives axonal transport of membrane cargoes to fulfill the metabolic needs of neurons and maintain synapses. We have previously demonstrated that kinesin-1, in addition to its well-established role in organelle transport, can drive MT-MT sliding by transporting "cargo" MTs along "track" MTs, resulting in dramatic cell shape changes. The mechanism and physiological relevance of this MT sliding are unclear. In addition to its motor domain, kinesin-1 contains a second MT-binding site, located at the C terminus of the heavy chain. Here, we mutated this C-terminal MT-binding site such that the ability of kinesin-1 to slide MTs is significantly compromised, whereas cargo transport is unaffected. We introduced this mutation into the genomic locus of kinesin-1 heavy chain (KHC), generating the Khc(mutA) allele. Khc(mutA) neurons displayed significant MT sliding defects while maintaining normal transport of many cargoes. Using this mutant, we demonstrated that MT sliding is required for axon and dendrite outgrowth in vivo. Consistent with these results, Khc(mutA) flies displayed severe locomotion and viability defects. To test the role of MT sliding further, we engineered a chimeric motor that actively slides MTs but cannot transport organelles. Activation of MT sliding in Khc(mutA) neurons using this chimeric motor rescued axon outgrowth in cultured neurons and in vivo, firmly establishing the role of sliding in axon outgrowth. These results demonstrate that MT sliding by kinesin-1 is an essential biological phenomenon required for neuronal morphogenesis and normal nervous system development.

Dendrite arborization requires the dynein cofactor NudE.

The microtubule-based molecular motor dynein is essential for proper neuronal morphogenesis. Dynein activity is regulated by cofactors, and the role(s) of these cofactors in shaping neuronal structure are still being elucidated. Using Drosophila melanogaster, we reveal that the loss of the dynein cofactor NudE results in abnormal dendrite arborization. Our data show that NudE associates with Golgi outposts, which mediate dendrite branching, suggesting that NudE normally influences dendrite patterning by regulating Golgi outpost transport. Neurons lacking NudE also have increased microtubule dynamics, reflecting a change in microtubule stability that is likely to also contribute to abnormal dendrite growth and branching. These defects in dendritogenesis are rescued by elevating levels of Lis1, another dynein cofactor that interacts with NudE as part of a tripartite complex. Our data further show that the NudE C-terminus is dispensable for dendrite morphogenesis and is likely to modulate NudE activity. We propose that a key function of NudE is to enhance an interaction between Lis1 and dynein that is crucial for motor activity and dendrite architecture.

Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains.

Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. Here we show that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. Our findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains.

In vivo optogenetic manipulations of endogenous proteins reveal spatiotemporal roles of microtubule and kinesin in dendrite patterning.

During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila. This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Intracellular transport: Finding the motor that will take you where you need to go.

The Golgi complex is a busy production hub. A new study reveals that a microtubule end-binding (EB) protein enriched at the trans-Golgi network in neurons is needed to pair dense core vesicles with a kinesin motor for transport to axons.

Live Imaging of Golgi Outposts in Drosophila Dendritic Arbors.

Neurons are polarized cells whose polarity and morphology rely on the robust localization of cellular organelles and cargo to axons or dendrites. Developing neurons require an active secretory pathway, which includes the endoplasmic reticulum and Golgi apparatus, to supply membrane and proteins to growing dendrites and axons. In some neurons, a subset of the Golgi called Golgi "outposts" localize to dendrites and contribute to local secretory networks. The movement and positioning of Golgi outposts have been correlated with dendrite branch growth and stabilization as the dendritic arbor is established. Live imaging is essential to capture the dynamic nature of these organelles. Here we outline a protocol to image and quantify Golgi outposts in peripheral sensory neurons in live, intact Drosophila larvae.

Introduction to CRISPR and Its Use in Drosophila.

The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics.

Generating CRISPR Alleles in Drosophila.

CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.

Light-induced trapping of endogenous proteins reveals spatiotemporal roles of microtubule and kinesin-1 in dendrite patterning of Drosophila sensory neurons.

Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Acetylated α-tubulin K394 regulates microtubule stability to shape the growth of axon terminals.

Microtubules are essential to neuron shape and function. Acetylation of tubulin has the potential to directly tune the behavior and function of microtubules in cells. Although proteomic studies have identified several acetylation sites in α-tubulin, the effects of acetylation at these sites remains largely unknown. This includes the highly conserved residue lysine 394 (K394), which is located at the αß-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons and reduces microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394R mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

Golgi Outposts Locally Regulate Microtubule Orientation in Neurons but Are Not Required for the Overall Polarity of the Dendritic Cytoskeleton.

Microtubule-organizing centers often play a central role in organizing the cellular microtubule networks that underlie cell function. In neurons, microtubules in axons and dendrites have distinct polarities. Dendrite-specific Golgi "outposts," in particular multicompartment outposts, have emerged as regulators of acentrosomal microtubule growth, raising the question of whether outposts contribute to establishing or maintaining the overall polarity of the dendritic microtubule cytoskeleton. Using a combination of genetic approaches and live imaging in a Drosophila model, we found that dendritic microtubule polarity is unaffected by eliminating known regulators of Golgi-dependent microtubule organization including the cis-Golgi matrix protein GM130, the fly AKAP450 ortholog pericentrin-like protein, and centrosomin. This indicates that Golgi outposts are not essential for the formation or maintenance of a dendrite-specific cytoskeleton. However, the overexpression of GM130, which promotes the formation of ectopic multicompartment units, is sufficient to alter dendritic microtubule polarity. Axonal microtubule polarity is similarly disrupted by the presence of ectopic multicompartment Golgi outposts. Notably, multicompartment outposts alter microtubule polarity independently of microtubule nucleation mediated by the γ-tubulin ring complex. Thus, although Golgi outposts are not essential to dendritic microtubule polarity, altering their organization correlates with changes to microtubule polarity. Based on these data, we propose that the organization of Golgi outposts is carefully regulated to ensure proper dendritic microtubule polarity.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.