Golgi Outposts Research Articles

γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

A distinct Golgi-targeting mechanism of dGM130 in Drosophila neurons.

GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. Here, we investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using in vivo imaging of Drosophila dendritic arborization (da) neurons. The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity.

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease.

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.

Primary neurons lacking the SNAREs vti1a and vti1b show altered neuronal development

BackgroundNeurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts.MethodsHippocampal and cortical neurons were isolated from Vti1a−/−Vti1b−/− double deficient, Vti1a−/−Vti1b+/−, Vti1a+/−Vti1b−/− and Vti1a+/−Vti1b+/− double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a−/−Vti1b−/− and Vti1a+/−Vti1b+/− control forebrains and analyzed by western blotting.ResultsGolgi outposts were present in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a−/−Vti1b−/− postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a+/−Vti1b+/−, but much more TrkB, which is the receptor for BDNF.ConclusionThe absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a−/−Vti1b−/− neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.

PP2A phosphatase regulates cell-type specific cytoskeletal organization to drive dendrite diversity.

Uncovering molecular mechanisms regulating dendritic diversification is essential to understanding the formation and modulation of functional neural circuitry. Transcription factors play critical roles in promoting dendritic diversity and here, we identify PP2A phosphatase function as a downstream effector of Cut-mediated transcriptional regulation of dendrite development. Mutant analyses of the PP2A catalytic subunit (mts) or the scaffolding subunit (PP2A-29B) reveal cell-type specific regulatory effects with the PP2A complex required to promote dendritic growth and branching in Drosophila Class IV (CIV) multidendritic (md) neurons, whereas in Class I (CI) md neurons, PP2A functions in restricting dendritic arborization. Cytoskeletal analyses reveal requirements for Mts in regulating microtubule stability/polarity and F-actin organization/dynamics. In CIV neurons, mts knockdown leads to reductions in dendritic localization of organelles including mitochondria and satellite Golgi outposts, while CI neurons show increased Golgi outpost trafficking along the dendritic arbor. Further, mts mutant neurons exhibit defects in neuronal polarity/compartmentalization. Finally, genetic interaction analyses suggest β-tubulin subunit 85D is a common PP2A target in CI and CIV neurons, while FoxO is a putative target in CI neurons.

Fringe-positive Golgi outposts unite temporal Furin 2 convertase activity and spatial Delta signal to promote dendritic branch retraction.

Golgi outposts (GOPs) in dendrites are known for their role in promoting branch extension, but whether GOPs have other functions is unclear. We found that terminal branches of Drosophila class IV dendritic arborization (C4da) neurons actively grow during the early third-instar (E3) larval stage but retract in the late third (L3) stage. Interestingly, the Fringe (Fng) glycosyltransferase localizes increasingly at GOPs in distal dendritic regionsthrough the E3 to the L3 stage. Expression of the endopeptidase Furin 2 (Fur2), which proteolyzes and inactivates Fng, decreases from E3 to L3 in C4da neurons, thereby increasing Fng-positive GOPs indendrites.The epidermal Delta ligand and neuronal Notch receptor, the substrate for Fng-mediated O-glycosylation, also negatively regulate dendrite growth. Fng inhibits actin dynamics in dendrites, linking dendritic branch retraction to suppression of the C4da-mediated thermal nociception response in late larval stages. Thus, Fng-positive GOPs function in dendrite retraction, which would add another function to the repertoire of GOPs in dendrite arborization.

Amyloid-β Impairs Dendritic Trafficking of Golgi-Like Organelles in the Early Phase Preceding Neurite Atrophy: Rescue by Mirtazapine.

Neurite atrophy with loss of neuronal polarity is a pathological hallmark of Alzheimer’s disease (AD) and other neurological disorders. While there is substantial agreement that disruption of intracellular vesicle trafficking is associated with axonal pathology in AD, comparatively less is known regarding its role in dendritic atrophy. This is a significant gap of knowledge because, unlike axons, dendrites are endowed with the complete endomembrane system comprising endoplasmic reticulum (ER), ER–Golgi intermediate compartment (ERGIC), Golgi apparatus, post-Golgi vesicles, and a recycling-degradative route. In this study, using live-imaging of pGOLT-expressing vesicles, indicative of Golgi outposts and satellites, we investigate how amyloid-β (Aβ) oligomers affect the trafficking of Golgi-like organelles in the different dendritic compartments of cultured rat hippocampal neurons. We found that short-term (4 h) treatment with Aβ led to a decrease in anterograde trafficking of Golgi vesicles in dendrites of both resting and stimulated (with 50 mM KCl) neurons. We also characterized the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi dynamics in dendrites. Mirtazapine treatment (10 μM) increased the number and both anterograde and retrograde motility, reducing the percentage of static Golgi vesicles. Finally, mirtazapine reverted the neurite atrophy induced by 24 h treatment with Aβ oligomers, suggesting that this drug is able to counteract the effects of Aβ by improving the dendritic trafficking of Golgi-related vesicles.

Sunday Driver Mediates Multi-Compartment Golgi Outposts Defects Induced by Amyloid Precursor Protein.

Golgi defects including Golgi fragmentation are pathological features of Alzheimer’s disease (AD). As a pathogenic factor in AD, amyloid precursor protein (APP) induces Golgi fragmentation in the soma. However, how APP regulates Golgi outposts (GOs) in dendrites remains unclear. Given that APP resides in and affects the movements of GOs, and in particular, reverses the distribution of multi-compartment GOs (mcGOs), we investigated the regulatory mechanism of mcGO movements in the Drosophila larvae. Knockdown experiments showed that the bidirectional mcGO movements were cooperatively controlled by the dynein heavy chain (Dhc) and kinesin heavy chain subunits. Notably, only Dhc mediated APP’s regulation of mcGO movements. Furthermore, by loss-of-function screening, the adaptor protein Sunday driver (Syd) was identified to mediate the APP-induced alteration of the direction of mcGO movements and dendritic defects. Collectively, by elucidating a model of bidirectional mcGO movements, we revealed the mechanism by which APP regulates the direction of mcGO movements. Our study therefore provides new insights into AD pathogenesis.

Microtubules, Myelin Sheaths, and Altered Behavior.

Mice are skttish creatures that innately hide from perceived threats by staying out of open areas, freezing to avoid predators, or hiding in dark spaces when an adverse stimulus is sensed. However, after inducing a global TPPP (tubulin polymerization promoting protein) gene knock-out (KO), which results in reduced myelin length and thickness (Fu et al., 2019), Nguyen et al. (2020) found that these natural behaviors become less pronounced (Fig. 1). They also report that fear conditioning is less effective in the Tppp KO mice, indicating that their ability to learn to fear a specific event is impaired. Fear impacts many brain circuits and is critical for survival, but the contribution of non-neuronal cells in fear circuits is not well understood. This work identifies a protein that influences both innate and learned fear via its role in microtubule nucleation in oligodendrocytes. Figure 1. A ) Normal branching and myelin sheath length in oligodendrocytes in WT mice is coupled with normal fear conditioning and innate fear responses. B ) Tppp KO results in highly branched oligodendrocytes with shorter, thinner myelin sheaths. These mice have dramatically reduced fear conditioning and innate fear responses. In the cell body of most cells, microtubules are nucleated from perinuclear centrosomes. These microtubule organizing centers (MTOCs) contain centrioles and a pericentriolar matrix containing g-tubulin ring complexes (γTURCs). γTURCs are the starting point for the initiation and subsequent polymerization of α/βtubulin dimers into cytoplasmic microtubules. Golgi outposts are a different type of MTOC that nucleate microtubules outside of the cell body in …

Local Secretory Trafficking Pathways in Neurons and the Role of Dendritic Golgi Outposts in Different Cell Models

A fundamental characteristic of neurons is the relationship between the architecture of the polarized neuron and synaptic transmission between neurons. Intracellular membrane trafficking is paramount to establish and maintain neuronal structure; perturbation in trafficking results in defects in neurodevelopment and neurological disorders. Given the physical distance from the cell body to the distal sites of the axon and dendrites, transport of newly synthesized membrane proteins from the central cell body to their functional destination at remote, distal sites represents a conundrum. With the identification of secretory organelles in dendrites, including endoplasmic reticulum (ER) and Golgi outposts (GOs), recent studies have proposed local protein synthesis and trafficking distinct from the conventional anterograde transport pathways of the cell body. A variety of different model organisms, including Drosophila, zebrafish, and rodents, have been used to probe the organization and function of the local neuronal secretory network. Here, we review the evidence for local secretory trafficking pathways in dendrites in a variety of cell-based neuronal systems and discuss both the similarities and differences in the organization and role of the local secretory organelles, especially the GOs. In addition, we identify the gaps in the current knowledge and the potential advances using human induced pluripotent stem cells (iPSCs) in defining local membrane protein trafficking in human neurons and in understanding the molecular basis of neurological diseases.

C9orf72-Associated Arginine-Rich Dipeptide Repeat Proteins Reduce the Number of Golgi Outposts and Dendritic Branches in Drosophila Neurons.

Altered dendritic morphology is frequently observed in various neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the cellular and molecular basis underlying these pathogenic dendritic abnormalities remains largely unclear. In this study, we investigated dendritic morphological defects caused by dipeptide repeat protein (DPR) toxicity associated with G4C2 expansion mutation of C9orf72 (the leading genetic cause of ALS and FTD) in Drosophila neurons and characterized the underlying pathogenic mechanisms. Among the five DPRs produced by repeat-associated non-ATG translation of G4C2 repeats, we found that arginine-rich DPRs (PR and GR) led to the most significant reduction in dendritic branches and plasma membrane (PM) supply in Class IV dendritic arborization (C4 da) neurons. Furthermore, expression of PR and GR reduced the number of Golgi outposts (GOPs) in dendrites. In Drosophila brains, expression of PR, but not GR, led to a significant reduction in the mRNA level of CrebA, a transcription factor regulating the formation of GOPs. Overexpressing CrebA in PR-expressing C4 da neurons mitigated PM supply defects and restored the number of GOPs, but the number of dendritic branches remained unchanged, suggesting that other molecules besides CrebA may be involved in dendritic branching. Taken together, our results provide valuable insight into the understanding of dendritic pathology associated with C9-ALS/FTD.

Fear Deficits in HypomyelinatedTpppKnock-Out Mice

Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to facilitate efficient electrical conduction. These myelin sheaths contain lamellar microtubules that enable vesicular transport into the inner sheath. Mechanistically, oligodendrocytes rely on Golgi outpost organelles and the associated protein tubulin polymerization promoting protein (TPPP) to nucleate or form new microtubules outside of the cell body. Consequently, elongation of lamellar microtubules is defective in Tppp knock-out (KO) mice, which have thinner and shorter myelin sheaths. We now explore the behavioral phenotypes of Tppp KO mice using a number of different assays. In open-field assays, Tppp KO mice display similar activity levels and movement patterns as wild-type mice, indicating that they do not display anxiety behavior. However, Tppp KO mice lack fear responses by two types of assays, traditional fear-conditioning assays and looming fear assays, which test for innate fear responses. Deficits in fear conditioning, which is a memory-dependent task, as well as in spatial memory tests, support possible short-term memory defects in Tppp KO mice. Together, our experiments indicate a connection between CNS myelination and behavioral deficits.

Golgi Outposts Nucleate Microtubules in Cells with Specialized Shapes.

Classically, animal cells nucleate or form new microtubules off the perinuclear centrosome. In recent years, the Golgi outpost has emerged as a satellite organelle that can function as an acentrosomal microtubule-organizing center (MTOC), nucleating new microtubules at distances far from the nucleus or cell body. Golgi outposts can nucleate new microtubules in specialized cells with unique cytoarchitectures, including Drosophila neurons, mouse muscle cells, and rodent oligodendrocytes. This review compares and contrasts topics of functional relevance, including Golgi outpost heterogeneity, formation and transport, as well as regulation of microtubule polarity and branching. Golgi outposts have also been implicated in the pathology of diseases including muscular dystrophy, and neurodegenerative diseases, such as Parkinson's disease (PD). Since Golgi outposts are relatively understudied, many outstanding questions regarding their function and roles in disease remain.

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.

Microtubule organization and dynamics in oligodendrocytes, astrocytes, and microglia.

Though much is known about microtubule organization and microtubule-based transport in neurons, the development and function of microtubules in glia are more enigmatic. In this review, we provide an overview of the literature on microtubules in ramified brain cells, including oligodendrocytes, astrocytes, and microglia. We focus on normal cell biology-how structure relates to function in these cells. In oligodendrocytes, microtubules are important for extension of processes that contact axons and for elongating the myelin sheath. Recent studies demonstrate that new microtubules can form outside of the oligodendrocyte cell body off of Golgi outpost organelles. In astrocytes and microglia, changes in cell shape and ramification can be influenced by neighboring cells and the extracellular milieu. Finally, we highlight key papers implicating glial microtubule defects in neurological injury and disease and discuss how microtubules may contribute to invasiveness in gliomas. Thus, future research on the mechanisms underlying microtubule organization in normal glial cell function may yield valuable insights on neurological disease pathology.

Predicting the Morphology of Class IV Neurons from the Dynamics of Dendritic Growth in Drosophila

The development of neurons into their stereotyped morphology is paramount to their function. The class IV neurons of the Drosophila melanogaster larva are sensory neurons that develop a complex, highly branched dendritic arbor sensitive to mechanical stimuli. The fully-developed dendritic tree results from a multitude of stochastic processes including dendritic tip growth, branching and self-avoidance. Previous studies have identified key molecular players involved in these processes, which include actin, microtubules, molecular motors, Golgi outposts, endosomes and the Down Syndrome cell adhesion molecule (DSCAM). However, it is yet unknown how these dendritic processes can produce the observed morphologies of the class IV neurons. Here, we formulated an agent-based model of dendritic growth that takes inputs from our previous measurements and analysis of the tip dynamics and branching process. The rules of the agent are based on the observed local behavior of dendritic tips: 1) Branches elongate stochastically by visiting three dynamical states, i.e, growing, paused and shrinking; 2) New branches are created at a rate that is proportional to the local density of dendrites; 3) Branches stop growing when they collide with another branch, which mimics contact-based retraction. Using these rules constrained by experimental observations, we show that the model recapitulates the morphology of class IV neurons. This allows us to bridge the scales of class IV dendritic growth by connecting the local tip dynamics (∼1 µm) to the global tree growth (∼1 mm). In summary, our results establish a mechanistic approach to understand how the small-scale growth dynamics of class IV neurons can shape the large-scale morphology of the dendritic tree.

The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for Myelination

Oligodendrocytes extend elaborate microtubule arbors that contact up to 50 axon segments per cell, then spiral around myelin sheaths, penetrating from outer to inner layers. However, how they establish this complex cytoarchitecture is unclear. Here, we show that oligodendrocytes contain Golgi outposts, an organelle that can function as an acentrosomal microtubule-organizing center (MTOC). We identify a specific marker for Golgi outposts-TPPP (tubulin polymerization promoting protein)-that we use to purify this organelle and characterize its proteome. In invitro cell-free assays, recombinant TPPP nucleates microtubules. Primary oligodendrocytes from Tppp knockout (KO) mice have aberrant microtubule branching, mixed microtubule polarity, and shorter myelin sheaths when cultured on 3-dimensional (3D) microfibers. Tppp KO mice exhibit hypomyelination with shorter, thinner myelin sheaths and motor coordination deficits. Together, our data demonstrate that microtubule nucleation outside the cell body at Golgi outposts by TPPP is critical for elongation of the myelin sheath.

A Microtubule-Myelination Connection.

Microtubules are critical for the extension of oligodendrocyte processes and myelin deposition, yet our knowledge of their microtubule biogenesis is limited. In this issue of Cell, Fu etal. (2019) identify an oligodendrocyte-enriched microtubule regulator that promotes microtubule growth from Golgi outposts and controls myelin sheath elongation, linking microtubule cytoarchitecture and myelination in the CNS.