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TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products ofTSNARE1and their cytoplasmic localization and function in endosomal trafficking in cortical neurons. We validated three primary isoforms ofTSNARE1expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Biochemical data demonstrate that tSNARE1 can compete with Stx12 for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE. Live-cell imaging in cortical neurons from mice of both sexes demonstrated that brain tSNARE1 isoforms localized to the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+late endosomes, and endogenous tSNARE1 displayed a similar localization in human neural progenitor cells and neuroblastoma cells. In mature rat neurons from both sexes, tSNARE1 localized to the dendritic shaft and dendritic spines, supporting a role for tSNARE1 at the postsynapse. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of an Myb-like domain, delayed the trafficking of the dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely by negatively regulating early endosomal to late endosomal trafficking.SIGNIFICANCE STATEMENTSchizophrenia is a severe and polygenic neuropsychiatric disorder. Understanding the functions of high-confidence candidate genes is critical toward understanding how their dysfunction contributes to schizophrenia pathogenesis.TSNARE1is one of the high-confidence candidate genes for schizophrenia risk, yet nothing was known about its cellular or physiological function. Here we describe the major isoforms ofTSNARE1and their cytoplasmic localization and function in the endosomal network in cortical neurons. Our results are consistent with the hypothesis that the majority of brain tSNARE1 acts as a negative regulator to endolysosomal trafficking.

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified neuronal putative TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high priority candidates was validated, including Myo16, Coro1A, SNAP47, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized TIRF microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated the RNAi-based knockdown of the unconventional myosin Myo16 in cortical neurons altered axonal branching patterns in a TRIM9 and netrin-1 dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function.

Schizophrenia is a severe and heritable neuropsychiatric disorder, which arises due to a combination of common genetic variation, rare loss of function variation, and copy number variation. Functional genomic evidence has been used to identify candidate genes affected by this variation, which revealed biological pathways that may be disrupted in schizophrenia. Understanding the contributions of these pathways are critical next steps in understanding schizophrenia pathogenesis. A number of genes involved in endocytosis are implicated in schizophrenia. In this review, we explore the history of endosomal trafficking in schizophrenia and highlight new endosomal candidate genes. We explore the function of these candidate genes and hypothesize how their dysfunction may contribute to schizophrenia.

The cerebellin family of proteins influences synapse formation and function. In this issue of PLOS Biology, Han and colleagues identify a new role for Cerebellin-1 in axon growth and guidance.

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the post-synaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of pre- synaptic cannabinoid receptor type 1 (CB1). The eCB system is well-situated to regulate neuronal network properties and coordinate pre- and post-synaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used western blot and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature (>DIV21) cultured rat cortical neurons, and used multielectrode array recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activities during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of oscillatory network activities.Significance statementNeurons are remarkably resilient to perturbations in network activities thanks to the expression of overlapping homeostatic adaptations. In response to network hyperactivity or silencing, neurons respond through regulating excitatory and inhibitory post-synaptic neurotransmitter receptors density, probability of pre-synaptic neurotransmitter release, and/or membrane excitability. The endocannabinoid system is a prominent signaling pathway at many synapses that is known to be involved in multiple forms of short- and long-term synaptic plasticity. Here we find that components of the endocannabinoid system are upregulated in response to chronic hyperexcitation of cultured cortical neurons, and that endocannabinoid signaling is required to maintain network activity but also suppresses network events during hyperexcitation. This work supports a novel tonic homeostatic function for the endocannabinoid system in neurons.

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak ΔF/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley's K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.

TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endolysosomal trafficking in murine cortical neurons. We validated four primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Live-cell imaging demonstrated that brain tSNARE1 isoforms localized to compartments of the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomal compartments. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of a Myb-like domain, delayed the trafficking of known endosomal cargo, Neep21, into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely through negatively regulating endocytic trafficking or maturation of early endosomes to late endosomes.

TRIM9 and TRIM67 are neuronally-enriched E3 ubiquitin. Both genes are required for neuronal morphological responses to the axon guidance cue netrin-1. For example, our previously published work demonstrated that the actin polymerase VASP and the netrin receptor DCC exhibit TRIM9 dependent ubiquitylation that is lost upon netrin stimulation. Deletion of either gene in the mouse results in subtle neuroanatomical anomalies yet overt deficits in spatial learning and memory. Despite their role in neuronal form and function, the identity of few TRIM9 or TRIM67 substrates are known. Here we performed ubiquitin remnant profiling approach in cultured wildtype and knockout murine embryonic cortical neurons to identify ubiquitylated peptides and proteins, with the ultimate goal of identifying substrates of TRIM9 and TRIM67 that exhibited reduced ubiquitylation in the absence of the ligase. This work reveals the ubiquitylome of developing cortical neurons.

The development of a functional nervous system entails establishing connectivity between appropriate synaptic partners. During axonal pathfinding, the developing axon navigates through the extracellular environment, extending toward postsynaptic targets. In the early 1900s, Ramon y Cajal suggested that the growth cone, a specialized, dynamic, and cytoskeletal-rich structure at the tip of the extending axon, is guided by chemical cues in the extracellular environment. A century of work supports this hypothesis and introduced myriad guidance cues and receptors that promote a variety of growth cone behaviors including extension, pause, collapse, retraction, turning, and branching. Here, we highlight research from the last two years regarding pathways implicated in axon pathfinding.

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.

Summary Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.

Boyer et al. describe reversible nondegradative ubiquitination of the actin polymerase VASP, mediated by a pair of E3 ubiquitin ligases TRIM9 and TRIM67. VASP regulation is necessary for responses of filopodia, growth cones, and axons to the guidance cue netrin-1., Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the filopodial actin polymerase VASP and consequently filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1–dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, filopodial stability, and axon guidance.

Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn.

Appropriate axon guidance is necessary to form accurate neuronal connections. Guidance cues stimulate reorganization of the cytoskeleton within the distal growth cone at the tip of the extending axon. Filopodia at the periphery of the growth cone have long been considered sensors for axon guidance cues, yet how they perceive and respond to extracellular cues remains ill-defined. Our previous work found that the filopodial actin polymerase VASP is regulated via TRIM9-dependent nondegradative ubiquitination, and that appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 antagonizes VASP ubiquitination by outcompeting the TRIM9:VASP interaction. This antagonistic role is required for netrin-1 dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model that coordinated regulation of nondegradative VASP ubiquitination by a pair of ligases is a critical element of axon guidance.

SUMMARY Neuronal morphogenesis involves dramatic plasma membrane expansion, fueled by soluble N-ethylmaleimide-sensitive factor attachment protein eceptors (SNARE)-mediated exocytosis. Distinct fusion modes described at synapses include full-vesicle fusion (FVF) and kiss-and-run fusion (KNR). During FVF, lumenal cargo is secreted and vesicle membrane incorporates into the plasma membrane. During KNR, a transient fusion pore secretes cargo but closes without membrane addition. In contrast, fusion modes are not described in developing neurons. Here, we resolve individual exocytic events in developing murine cortical neurons and use classification tools to identify four distinguishable fusion modes: two FVF-like modes that insert membrane material and two KNR-like modes that do not. Discrete fluorescence profiles suggest distinct behavior of the fusion pore. Simulations and experiments agree that FVF-like exocytosis provides sufficient membrane material for morphogenesis. We find the E3 ubiquitin ligase TRIM67 promotes FVF-like exocytosis in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes and, thus, SNAP47 involvement in exocytosis., Graphical Abstract, In Brief Urbina et al. identify four exocytic modes in developing neurons: KNRd, KNRi, FVFd, FVFi. Simulations and experiments suggest that FVFi and FVFd provide material for plasma membrane expansion. Deletion of Trim67 decreases FVFi and FVFd while reducing surface area. SNAP47 incorporation into SNARE complexes alters fusion pore behavior, increasing KNRd.

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. This article focuses on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1293-1307, 2016.

During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neuronsin vitroandin vivo. Embryonic hippocampal and adult-born dentate granule neurons lackingTrim9exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered byTrim9deletion, a significant number ofTrim9−/−adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons inTrim9−/−mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors.SIGNIFICANCE STATEMENTAppropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion ofTrim9elevated dendritic arborization of hippocampal neuronsin vitroandin vivo. Adult-born dentate granule cells lackingTrim9similarly exhibited excessive dendritic arborization and mislocalization of cell bodiesin vivo. These cellular defects were associated with severe deficits in spatial learning and memory.

Proper neuronal wiring is central to all bodily functions, sensory perception, cognition, memory, and learning. Establishment of a functional neuronal circuit is a highly regulated and dynamic process involving axonal and dendritic branching and navigation toward appropriate targets and connection partners. This intricate circuitry includes axo-dendritic synapse formation, synaptic connections formed with effector cells, and extensive dendritic arborization that function to receive and transmit mechanical and chemical sensory inputs. Such complexity is primarily achieved by extensive axonal and dendritic branch formation and pruning. Fundamental to neuronal branching are cytoskeletal dynamics and plasma membrane expansion, both of which are regulated via numerous extracellular and intracellular signaling mechanisms and molecules. This review focuses on recent advances in understanding the biology of neuronal branching.

Understanding how axons are guided to target locations within the brain is of fundamental importance for neuroscience, and is a widely studied area of research. Biologists have an unmet need for reliable and easily accessible methods that generate stable, soluble molecular gradients for the investigation of axon guidance. Here we developed a microfluidic device with contiguous media-filled compartments that uses gravity-driven flow to generate a stable and highly reproducible gradient within a viewing compartment only accessible to axons. This device uses high-resistance microgrooves to both direct the growth of axons into an isolated region and to generate a stable gradient within the fluidically isolated axon viewing compartment for over 22 h. Establishing a stable gradient relies on a simple and quick pipetting procedure with no external pump or tubing. Since the axons extend into the axonal compartment through aligned micro-grooves, the analysis of turning is simplified. Further, the multiple microgrooves in parallel alignment serve to increase sample sizes, improving statistical analyses. We used this method to examine growth cone turning in response to the secreted axon guidance cue netrin-1. We report the novel finding that growth cones of embryonic mouse cortical axons exhibited attractive turning in the lower concentrations of netrin-1, but were repulsed when exposed to higher concentrations. We also performed immunocytochemistry in growth cones exposed to a netrin-1 gradient within the axon viewing compartment and show that netrin receptors associated with both attraction and repulsion, DCC and UNC5H, localized to these growth cones. Together, we developed an accessible gradient chamber for higher throughput axon guidance studies and demonstrated its capabilities.

Specific class I members of the TRIM family of E3 ubiquitin ligases have been implicated in neuronal development from invertebrates to mammals. The single invertebrate class I TRIM and mammalian TRIM9 regulate axon branching and guidance in response to the axon guidance cue netrin-1, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Optiz Syndrome, characterized by midline defects and often mild intellectual disability. TRIM67 is the most evolutionarily conserved vertebrate class I TRIM, yet is the least studied. Here we show that TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC, and is differentially enriched in specific brain regions at specific developmental points. We describe the anatomical and behavioral consequences of deletion of murine Trim67. While viable, mice lacking Trim67 display severe impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function and sensorimotor gating. Additionally, they exhibit abnormal anatomy of several brain regions, including the hippocampus, striatum and thalamus, as well as the corpus callosum. This study demonstrates the necessity for TRIM67 in appropriate brain development and function.SIGNIFICANCE STATEMENTAs a family, class I TRIM E3 ubiquitin ligases play important roles in neuronal development and function, potentially cooperatively. TRIM67 is the most evolutionarily conserved class I TRIM and is developmentally regulated and brain-enriched. Deletion of murine Trim67 results in malformations of a subset subcortical brain regions and of cortical and subcortical myelinated fiber tracts, as well as deficits in spatial memory, motor function, sociability and sensorimotor gating. We conclude that TRIM67 is critical for appropriate brain development and behavior, potentially downstream of the axon guidance cue netrin, and in cooperation with class I TRIM9.

Urbina et al. use a new computer-vision image analysis tool and extended clustering statistics to demonstrate that the spatiotemporal distribution of constitutive VAMP2-mediated exocytosis is dynamic in developing neurons. The exocytosis pattern is modified by both developmental time and the guidance cue netrin-1, regulated differentially in the soma and neurites, and distinct from exocytosis in nonneuronal cells., Neurite elongation and branching in developing neurons requires plasmalemma expansion, hypothesized to occur primarily via exocytosis. We posited that exocytosis in developing neurons and nonneuronal cells would exhibit distinct spatiotemporal organization. We exploited total internal reflection fluorescence microscopy to image vesicle-associated membrane protein (VAMP)–pHluorin—mediated exocytosis in mouse embryonic cortical neurons and interphase melanoma cells, and developed computer-vision software and statistical tools to uncover spatiotemporal aspects of exocytosis. Vesicle fusion behavior differed between vesicle types, cell types, developmental stages, and extracellular environments. Experiment-based mathematical calculations indicated that VAMP2-mediated vesicle fusion supplied excess material for the plasma membrane expansion that occurred early in neuronal morphogenesis, which was balanced by clathrin-mediated endocytosis. Spatial statistics uncovered distinct spatiotemporal regulation of exocytosis in the soma and neurites of developing neurons that was modulated by developmental stage, exposure to the guidance cue netrin-1, and the brain-enriched ubiquitin ligase tripartite motif 9. In melanoma cells, exocytosis occurred less frequently, with distinct spatial clustering patterns.

Morphological elongation of developing neurons requires plasmalemma expansion, hypothesized to occur primarily via exocytosis. We posited that exocytosis in developing neurons and non-neuronal cells exhibit distinct spatiotemporal organization. We exploited TIRF microscopy to image VAMP-pHluorin mediated exocytosis in murine embryonic cortical neurons and interphase melanoma cells, and developed computer-vision software and statistical tools to uncover spatiotemporal aspects of exocytosis. Vesicle fusion behavior differed between vesicle types, cell types, developmental stage, and extracellular environment. Experiment-based mathematical calculations indicated that VAMP2-mediated vesicle fusion supplied excess material for the plasma membrane expansion that occurred early in neuronal morphogenesis, which was balanced in part by clathrin-mediated endocytosis. Spatial statistics uncovered distinct spatiotemporal regulation of exocytosis in the soma and neurites of developing neurons that was modulated by developmental stage, exposure to the guidance cue netrin-1, and the brain-enriched ubiquitin ligase TRIM9. In melanoma cells, exocytosis occurred less frequently with distinct spatial clustering patterns.

In the presence of netrin, tripartite motif protein 9 (TRIM9) promotes deleted in colorectal cancer (DCC) clustering, but TRIM9-dependent ubiquitination of DCC is reduced. Loss of ubiquitination promotes an interaction between DCC and FAK and FAK activation. FAK activation is required for the progression from SNARE assembly to exocytic vesicle fusion, which supplies membrane material for axon branching., Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.

SUMMARY Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered β-transducin repeat-containing protein (β-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking IκBα degradation and thereby dampening nuclear factor κB (NF-κB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-κB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target., Graphical Abstract, In Brief Neuroinflammation drives pathology during brain injury. Zeng et al. show that TRIM9 is induced after ischemic insults in young mice, but not old mice, and promotes resolution of neuroinflammation. AAV-mediated TRIM9 therapy in aged mice restricts neuroinflammation and alleviates stroke damage, representing a potential therapeutic target for brain injury.

During development, neurons progress through rapid yet stereotypical shape changes to achieve proper neuronal connectivity. This morphological progression requires carefully orchestrated plasma membrane expansion, insertion of membrane components including receptors for extracellular cues into the plasma membrane and removal and trafficking of membrane materials and proteins to specific locations. This review outlines the cellular machinery of membrane trafficking that play an integral role in neuronal cell shape change and function from initial neurite formation to pathway navigation and synaptogenesis.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein is a multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development.

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins., National Institutes of Health (U.S.) (Grant GM58801), National Cancer Institute (U.S.) (Grant U54-CA112967), Howard Hughes Medical Institute

Class I members of the tripartite motif (TRIM) family of E3 ubiquitin ligases evolutionarily appeared just prior to the advent of neuronal like cells and have been implicated in neuronal development from invertebrates to mammals. The single Class I TRIM inDrosophila melanogasterandCaenorhabditis elegansand the mammalian Class I TRIM9 regulate axon branching and guidance in response to the guidance cue netrin, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Opitz Syndrome, characterized by midline defects and often intellectual disability. We find that although TRIM67 is the least studied vertebrate Class I TRIM, it is the most evolutionarily conserved. Here we show that mammalian TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC and is differentially enriched in specific brain regions during development and adulthood. We describe the anatomical and behavioral consequences of deletion of murineTrim67. While viable, mice lackingTrim67exhibit abnormal anatomy of specific brain regions, including hypotrophy of the hippocampus, striatum, amygdala, and thalamus, and thinning of forebrain commissures. Additionally,Trim67−/−mice display impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function, and sensorimotor gating, whereas several other behaviors remain intact. This study demonstrates the necessity for TRIM67 in appropriate brain development and behavior.

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Cell migration requires spatial and temporal regulation of filamentous actin (F-actin) dynamics. This regulation is achieved by distinct actin-associated proteins, which mediate polymerization, depolymerization, severing, contraction, bundling or engagement to the membrane. Mammalian Diaphanous-related (mDia) formins, which nucleate, processively elongate, and in some cases bundle actin filaments, have been extensively studied in vitro, but their function in the cell has been less well characterized. Here we study the role of mDia2 activity in the dynamic organization of F-actin in migrating epithelial cells. We find that mDia2 localizes in the lamella of migrating epithelial cells, where it is involved in the formation of a stable pool of cortical actin and in maintenance of polymerization-competent free filament barbed ends at focal adhesions. Specific inhibition of mDia2 alters focal adhesion turnover and reduces migration velocity. We suggest that the regulation of filament assembly dynamics at focal adhesions may be necessary for the formation of a stable pool of cortical lamella actin and the proper assembly and disassembly dynamics of focal adhesions, making mDia2 an important factor in epithelial cell migration.

SummaryNeuronal growth cone filopodia contain guidance receptors and contribute to axon guidance; however, the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here, we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine Trim9+/+ and Trim9−/− cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. Trim9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments, respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells.

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin–binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle αTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Background: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, [1]), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. Results: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. Conclusions: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.

Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)–deficient MV[superscript D7] fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2[superscript M/A] rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function., National Institutes of Health (U.S.) (Grant GM58801), National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1-U54-CA112967)

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion., American Heart Association (Fellowship 0615692T), National Institutes of Health (U.S.) (Grant GM68678)

Automated image analysis of unperturbed cells reveals a new sequence of events underlying protrusion of the cell membrane.

Neurons establish their unique morphology by elaborating multiple neurites that subsequently form axons and dendrites. Neurite initiation entails significant surface area expansion, necessitating addition to the plasma membrane. We report that regulated membrane delivery coordinated with the actin cytoskeleton is crucial for neuritogenesis, and identify two independent pathways that use distinct exocytic and cytoskeletal machinery to drive neuritogenesis. One pathway employs Ena/VASP-regulated actin dynamics coordinated with VAMP2-mediated exocytosis, and involves a novel role for Ena/VASP in exocytosis. A second mechanism occurs in the presence of laminin through integrin-dependent activation of FAK and src, and utilizes coordinated activity of the Arp2/3 complex and VAMP7-mediated exocytosis. We conclude that neuritogenesis can be driven by two distinct pathways that differentially coordinate cytoskeletal dynamics and exocytosis. These regulated changes and coordination of cytoskeletal and exocytic machinery may be utilized in other physiological contexts involving cell motility and morphogenesis.

Filopodia are actin-based structures composed of parallel bundles of actin filaments and various actin-associated proteins, and they play important roles in cell-cell signaling, guidance toward chemoattractants, and adhesion to the extracellular matrix. Two mechanisms for the formation of filopodia have been suggested, each using different sets of actin-regulating proteins, creating some controversy in the field. New molecules, some of unknown functions, have also been implicated in filopodium formation, suggesting that other possible mechanisms of filopodium formation exist. We discuss established and novel proteins that mediate the formation and dynamics of filopodia, different mechanisms of filopodium formation, and the various functions that distinct filopodia perform.