Biology subjects

Wildonger, J.

Publications and source records attributed to Wildonger, J..

9 recordsLinked to original sources

bioRxiv2025

Mutating different α-tubulin acetylation sites has distinct effects on axon terminal morphogenesis in Drosophila melanogaster

Microtubules are created from uniform - and {beta}-tubulin building blocks but typically carry out a variety of specialized functions within a cell. The post-translational modification of tubulin is one means by which microtubule function can be tuned to match different cellular activities. While multiple sites of acetylation have been identified in tubulin, particularly -tubulin, the effect of acetylation at different sites on microtubule function remains poorly characterized. Here, we took a genetic approach in Drosophila to disrupt three conserved sites of acetylation (K326, K370, K401) in endogenous -tubulin and characterized the effects on neuronal development. Acetylation-blocking mutagenesis of -tubulin K326 (K326A) perturbed larval locomotion and reduced axon terminal growth at the neuromuscular junction. These deficits were accompanied by a reduction in stable microtubules, suggesting that the -tubulin K326A mutation exerts its effect by disrupting microtubule stability. In contrast, mutagenesis of -tubulin K370 and K401 had virtually no effect on microtubule stability, suggesting that the effects of these mutations on axon terminal morphogenesis and survival may be mediated through a different mechanism. Altogether, the varied effects of these mutations suggests that acetylation at these three different sites may regulate different aspects of microtubule function within developing neurons.

cell biology↗

bioRxiv2025

ACETYLATED α-TUBULIN LYSINE 394 IS CRITICAL FOR AXON OUTGROWTH IN THE ADULT MUSHROOM BODIES OF DROSOPHILA MELANOGASTER

Disruptions in the microtubule cytoskeleton play a role in various neurological diseases that afflict a large fraction of the population. Microtubule function is regulated by post-translational modifications like acetylation, and one consistently identified acetylation site in mammals and Drosophila melanogaster is -tubulin lysine 394 (K394). Our previous research demonstrated that an acetylation-blocking point mutation--K394R--causes a decrease in microtubule stability in axon terminals at the developing neuromuscular junction. Here, we asked whether K394 acetylation regulates the development of additional neuronal structures. Using the central brain mushroom body as a model, we found that K394R results in {beta} lobe overextension at the midline. The K394R phenotype manifests during metamorphosis and affects {beta} lobe growth in a cell-autonomous manner. Our data suggest that the K394R phenotype may result from changes in Tau, a microtubule-associated protein enriched in the mushroom body and known to play a critical role in regulating neuronal microtubules. Knocking-out tau resulted in defects in midline crossing similar to K394R. However, when the loss of tau was combined with K394R, {beta} lobe extension was normal--indicating that the loss of tau suppresses the K394R phenotype and vice versa. While overexpressing tau also resulted in a midline crossing phenotype, K394R in combination with elevated Tau resulted in a severely malformed mushroom body. Altogether, our work suggests that K394R interacts with tau to regulate axon outgrowth during mushroom body development and raises the potential of manipulating K394 acetylation to ameliorate neurological disease resulting from axonal growth defects and changes in Tau.

cell biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2023

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.

biophysics↗

bioRxiv2022

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 delivery of sensory ion channels to the dendritic membrane. However, it is not well understood how ion channels are trafficked to sensory dendrites and whether their delivery is coordinated with dendrite growth. We investigated the trafficking of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in peripheral sensory neurons in fruit fly larvae. We used CRISPR-Cas9 genome engineering to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Strikingly, Ppk1 is present throughout the membrane of actively growing dendrites, and Ppk1 density scales in proportion to the dendritic membrane, even when dynein-mediated transport to dendrites is disrupted. Our data suggest that Ppk1 is integral to the membrane of growing dendrites and implicate the recycling endosome GTPase Rab11 in the forward trafficking of Ppk1 to dendrites. Together, our results suggest that Ppk channel transport is coordinated with dendrite morphogenesis, thus ensuring proper ion channel levels and distribution in sensory dendrites. Author SummaryPeripheral sensory neurons are essential for an organism to interact with its environment. Neurons are composed of signal-receiving dendrites and a signal-sending axon. Ion channels distributed throughout sensory dendrites transduce external stimuli into chemical signals, however the mechanisms that localize ion channels to sensory dendrites are not well understood. Both the composition of ion channels in the dendrites and the structure of a sensory neurons dendritic arbor are important for how it functions to sense external stimuli. Using live imaging and genomic engineering, we have discovered that the localization of a sensory ion channel, Pickpocket, in fruit fly sensory neurons is coordinated with growth of the dendritic arbor and that Pickpocket levels scale in proportion to dendrite length, even when transport to dendrites is disrupted. We also developed a novel genetically encoded approach to visualize the membrane expression of proteins in a living organism utilizing the split-GFP system. We found that both the secretory and endosomal networks mediate the forward trafficking of Pickpocket during neuronal morphogenesis, thus coordinating membrane growth with ion channel delivery. Our findings reveal that actively growing sensory dendrites are equipped with the ion channels needed for sensing external stimuli.

neuroscience↗

bioRxiv2021

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

Microtubules are essential to neuron shape and function. Therefore, the stability of the microtubule cytoskeleton must be carefully regulated. Acetylation of tubulin has the potential to directly tune microtubule stability, and proteomic studies have identified several acetylation sites in -tubulin. This includes the highly conserved residue lysine 394 (K394), which is located at the {beta}-tubulin dimer interface. Using a fly model, we show that -tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous -tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons by reducing 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 K394 mutant. Thus, our findings implicate -tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

cell biology↗

bioRxiv2019

Golgi outposts locally regulate microtubule orientation in neurons but are not required for the overall polarity of the dendritic cytoskeleton

Microtubule-organizing centers (MTOCs) 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 multi-compartment outposts, have emerged as regulators of acentrosomal microtubule growth, raising the question of whether outposts contribute to establishing the overall polarity of the dendritic microtubule cytoskeleton. The cis-Golgi matrix protein GM130 has roles in both the MTOC activity of Golgi and in connecting Golgi compartments to form multi-compartment units. Using a combination of genetic approaches and live imaging in a Drosophila model, we found that GM130 is not essential for the overall polarity of the dendritic microtubule cytoskeleton. However, the mislocalization of multi-compartment Golgi outposts to axons disrupts the uniform orientation of axonal microtubules. This suggests that outposts have the capacity to influence microtubule polarity and, as our data indicate, likely do so independently of microtubule nucleation mediated by the {gamma}-tubulin ring complex ({gamma}-TuRC). Altogether, our results are consistent with the model that multi-compartment Golgi outposts may locally influence microtubule polarity, but that outposts are not necessary for the overall polarity of the dendritic microtubule cytoskeleton.

cell biology↗

bioRxiv2018

Autoinhibition of kinesin-1 is essential to the dendrite-specific localization of Golgi outposts

Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure-function analysis of endogenous Drosophila kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.\n\nSummaryNeuronal polarity relies on the axon-or dendrite-specific localization of cargo by molecular motors such as kinesin-1. These studies show autoinhibition regulates both kinesin-1 activity and localization to keep dendritic cargo from entering axons.

cell biology↗

bioRxiv2017

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

Microtubules are essential to 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 -tubulin lysine 40 (K40) is a prominent, 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 -tubulin in vivo using a new fly strain that facilitates the rapid knock-in of designer -tubulin alleles. 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 -tubulin K40. Mutagenesis of K40 reveals moderate yet significant changes in dendritic lysosome transport, microtubule polymerization, and Futsch distribution in dendrites but not axons. Our studies point to an unappreciated role for -tubulin K40 and acetylation in dendrite morphogenesis. While our results are consistent with the idea that microtubule acetylation patterns microtubule function within neurons, they also suggest there may be a structural requirement for -tubulin K40.\n\nSummary StatementNeurons are enriched in post-translationally modified microtubules. Targeted mutagenesis of endogenous -tubulin in flies reveals that dendrite branch refinement is altered by acetylation-blocking mutations.

cell biology↗