RESUMO
The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.
Assuntos
Degrons , Ubiquitina , Ubiquitina/metabolismo , Proteínas/metabolismo , Ubiquitinação , Peptídeos/metabolismoRESUMO
cAMP is a ubiquitous second messenger with many functions in diverse organisms. Current cAMP sensors, including Föster resonance energy transfer (FRET)-based and single-wavelength-based sensors, allow for real time visualization of this small molecule in cultured cells and in some cases in vivo. Nonetheless the observation of cAMP in living animals is still difficult, typically requiring specialized microscopes and ex vivo tissue processing. Here we used ligand-dependent protein stabilization to create a new cAMP sensor. This sensor allows specific and sensitive detection of cAMP in living zebrafish embryos, which may enable new understanding of the functions of cAMP in living vertebrates.
Assuntos
Técnicas Biossensoriais , AMP Cíclico , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ligantes , Peixe-Zebra/metabolismoRESUMO
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.
Assuntos
Dasatinibe/farmacologia , Epigênese Genética , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigenoma , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Memória Imunológica , Ativação Linfocitária , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Neoplasias Experimentais/terapia , Domínios Proteicos , Estabilidade Proteica , Receptores de Antígenos Quiméricos/química , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais , Linfócitos T/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Here, we report a method to regulate cellular protein levels by introducing a ubiquitin variant between a destabilizing domain (DD) and the regulated protein. When produced in the absence of a stabilizing ligand the DD dominates and the entire fusion protein is processively degraded by the proteasome. In the presence of the stabilizing ligand the fusion protein is metabolically stable and becomes a substrate for abundant ubiquitin-specific proteases, liberating a native, or a near-native protein-of-interest. This technique is thus particularly useful for the study of proteins whose free N terminus is required for proper function. In addition, removal of the DD in the presence of stabilizing ligand leads to higher expression levels of regulated protein when cells experience transient exposure to a stabilizing ligand, such as in a living animal receiving a single dose of a pharmacological agent as the stabilizing ligand.
Assuntos
Engenharia de Proteínas/métodos , Ubiquitina/metabolismo , Animais , Peptídeo Hidrolases/metabolismo , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/química , Ubiquitina/genéticaRESUMO
Tools that can directly regulate the activity of any protein-of-interest are valuable in the study of complex biological processes. Herein, we describe the development of a novel protein domain that exhibits small molecule-dependent stability and fluorescence based on the bilirubin-inducible fluorescent protein, UnaG. When genetically fused to any protein-of-interest, this fluorescent destabilizing domain (FDD) confers its instability to the entire fusion protein, facilitating the rapid degradation of the fusion. In the presence of its cognate ligand bilirubin (BR), the FDD fusion becomes stable and fluorescent. This new chemical genetic tool allows for rapid, reversible, and tunable control over the stability and fluorescence of a wide range of protein targets.
Assuntos
Corantes Fluorescentes/química , Animais , Camundongos , Microscopia de Fluorescência , Células NIH 3T3RESUMO
The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Células NIH 3T3 , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/genética , Imagem com Lapso de Tempo/métodosRESUMO
Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called 'destabilizing domains', whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.
Assuntos
Fenômenos Fisiológicos Celulares , Regulação da Expressão Gênica , Transdução de Sinais , Transcrição Gênica , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , CamundongosRESUMO
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gß subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.
Assuntos
Quimiotaxia , Raios Ultravioleta , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Equipamento , Fluoresceínas/análise , Corantes Fluorescentes/análise , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Leucemia Mieloide Aguda/patologia , Microscopia de Fluorescência/métodos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas de Neoplasias/fisiologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fotoquímica , RNA Interferente Pequeno/farmacologia , Imagem com Lapso de TempoRESUMO
A common strategy to understand a biological system is to selectively perturb it and observe its response. Although technologies now exist to manipulate cellular systems at the genetic and transcript level, the direct manipulation of functions at the protein level can offer significant advantages in precision, speed, and reversibility. Combining the specificity of genetic manipulation and the spatiotemporal resolution of light- and small molecule-based approaches now allows exquisite control over biological systems to subtly perturb a system of interest in vitro and in vivo. Conditional perturbation mechanisms may be broadly characterized by change in intracellular localization, intramolecular activation, or degradation of a protein-of-interest. Here we review recent advances in technologies for conditional regulation of protein function and suggest further areas of potential development.
Assuntos
Proteínas/metabolismo , Regulação Alostérica , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas/química , Proteínas/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.
Assuntos
Avena/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Fotólise , Fototropinas/química , Fototropinas/genética , Estabilidade Proteica , Animais , Avena/química , Luz , Camundongos , Células NIH 3T3 , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Peixe-ZebraRESUMO
To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.
Assuntos
Dobramento de Proteína , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Benzoquinonas/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
Acute manipulation of gene and protein function in the brain is essential for understanding the mechanisms of nervous system development, plasticity and information processing. Here we describe a technique based on a destabilized Cre recombinase (DD-Cre) whose activity is controlled by the antibiotic trimethoprim (TMP). We show that DD-Cre triggers rapid TMP-dependent recombination of loxP-flanked ('floxed') alleles in mouse neurons in vivo and validate the use of this system for neurobehavioral research.
Assuntos
Regulação da Expressão Gênica , Integrases/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Recombinação Genética/efeitos dos fármacos , Trimetoprima/farmacologiaRESUMO
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Estabilidade Proteica , Animais , Camundongos , Células NIH 3T3 , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Endoteliais/fisiologia , Hipertensão Pulmonar/tratamento farmacológico , Tacrolimo/farmacologia , Animais , Apoptose , Proteína Morfogenética Óssea 4/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microvasos/patologia , Neointima/tratamento farmacológico , Neointima/metabolismo , Neointima/patologia , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Neoplasias da Mama/etiologia , Recidiva Local de Neoplasia/etiologia , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Miosinas Cardíacas/metabolismo , Proteínas Quinases Associadas com Morte Celular , Regulação para Baixo , Feminino , Humanos , Camundongos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Receptor ErbB-2/análise , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.
Assuntos
Comunicação Celular/imunologia , Genes Reporter , Vigilância Imunológica , Neoplasias Mamárias Experimentais/imunologia , Modelos Biológicos , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologiaRESUMO
Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Axônios/metabolismo , Dendritos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Axônios/ultraestrutura , Células Cultivadas , Dendritos/ultraestrutura , Microscopia Eletrônica de Varredura , Transporte Proteico/fisiologia , RatosRESUMO
The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.
Assuntos
Espaço Intracelular/metabolismo , Proteínas/química , Proteínas/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
In neurons, transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here, we use a novel pulse-chase system, which allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum to follow movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon, they very rarely moved beyond the axon initial segment but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.