RESUMO
The cortical actin network in neurosecretory cells is a dense mesh of actin filaments underlying the plasma membrane. Interaction of actomyosin with vesicular membranes or the plasma membrane is vital for tethering, retention, transport as well as fusion and fission of exo- and endocytic membrane structures. During regulated exocytosis the cortical actin network undergoes dramatic changes in morphology to accommodate vesicle docking, fusion and replenishment. Most of these processes involve plasma membrane Phosphoinositides (PIP) and investigating the interactions between the actin cortex and secretory structures has become a hotbed for research in recent years. Actin remodelling leads to filopodia outgrowth and the creation of new fusion sites in neurosecretory cells and actin, myosin and dynamin actively shape and maintain the fusion pore of secretory vesicles. Changes in viscoelastic properties of the actin cortex can facilitate vesicular transport and lead to docking and priming of vesicle at the plasma membrane. Small GTPase actin mediators control the state of the cortical actin network and influence vesicular access to their docking and fusion sites. These changes potentially affect membrane properties such as tension and fluidity as well as the mobility of embedded proteins and could influence the processes leading to both exo- and endocytosis. Here we discuss the multitudes of actin and membrane interactions that control successive steps underpinning regulated exocytosis.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , HumanosRESUMO
In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI.
Assuntos
Actinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exocitose/fisiologia , Vesículas Secretórias/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células PC12 , Fosfoproteínas/metabolismo , RatosRESUMO
Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells.
Assuntos
Actinas/metabolismo , Células Cromafins/fisiologia , Células Cromafins/ultraestrutura , Endocitose/fisiologia , Endossomos/metabolismo , Miosina Tipo II/metabolismo , Glândulas Suprarrenais/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Dextranos/metabolismo , Dinaminas/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Hidrazonas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Miosina Tipo II/antagonistas & inibidores , Naftóis/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Rodaminas/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.
Assuntos
Autofagia/efeitos dos fármacos , Toxinas Botulínicas Tipo A/metabolismo , Hipocampo/citologia , Neurônios/citologia , Neurotoxinas/metabolismo , Androstadienos/farmacologia , Animais , Animais Recém-Nascidos , Autofagia/fisiologia , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Toxinas Botulínicas Tipo A/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas In Vitro , Macrolídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Neurotoxinas/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptores de Fator de Crescimento Neural/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , WortmaninaRESUMO
Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1(Δ317-333)) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1(Δ317-333) to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1(Δ317-333) binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1(Δ317-333) could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.
Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Proteínas Munc18/metabolismo , Sintaxina 1/metabolismo , Animais , Membrana Celular/genética , Humanos , Proteínas Munc18/genética , Células PC12 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Ratos , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sintaxina 1/genéticaRESUMO
Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.
Assuntos
Sistemas Neurossecretores/metabolismo , Pseudópodes/metabolismo , Actinas/metabolismo , Actomiosina/antagonistas & inibidores , Actomiosina/metabolismo , Animais , Bovinos , Fusão Celular , Células Cultivadas , Células Cromafins/fisiologia , Células Cromafins/ultraestrutura , Vesículas Citoplasmáticas/fisiologia , Vesículas Citoplasmáticas/ultraestrutura , Citoesqueleto/fisiologia , Exocitose/fisiologia , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo II/fisiologia , Plasticidade Neuronal/fisiologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/efeitos dos fármacos , Polimerização , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Vesículas Secretórias/fisiologia , Vesículas Secretórias/ultraestruturaRESUMO
Most mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study, we reveal that the speed of various large cargoes undergoing axonal transport is significantly slower than that of small ones and that the transit of diverse-sized cargoes causes an acute, albeit transient, axonal radial expansion, which is immediately restored by constitutive axonal contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity significantly increases axon diameter by detaching the NM-II from F-actin and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged NM-II inactivation leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actomiosina/genética , Autofagossomos/ultraestrutura , Axônios/ultraestrutura , Neurônios/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Autofagossomos/genética , Transporte Axonal/genética , Axônios/metabolismo , Movimento Celular/genética , Proteínas Contráteis/genética , Cones de Crescimento/metabolismo , Microtúbulos/genética , Microtúbulos/ultraestrutura , Contração Muscular/genética , Neurônios/ultraestrutura , Transporte Proteico/genética , RatosRESUMO
This corrects the article DOI: 10.1038/ncomms13660.
RESUMO
Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transmissão Sináptica , Sintaxina 1/genética , Animais , Sítios de Ligação , Difusão , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Regulação da Expressão Gênica , Larva/citologia , Larva/fisiologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Imagem Molecular/métodos , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Optogenética , Ligação Proteica , Transporte Proteico , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Sintaxina 1/metabolismo , Toxina Tetânica/genética , Toxina Tetânica/metabolismoRESUMO
Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle "pools" are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Organelas/metabolismo , Vesículas Secretórias/metabolismo , Animais , Transporte Biológico , Células PC12 , Ratos , Proteínas SNARE/metabolismoRESUMO
Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Neurônios Motores/metabolismo , Neurotoxinas/farmacologia , Vesículas Sinápticas/metabolismo , Animais , Exocitose/efeitos dos fármacos , Exocitose/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Neurônios Motores/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Transporte Proteico/genética , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1(Δ317-333)) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1(WT) mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1(Δ317-333), suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.
Assuntos
Proteínas Munc18/química , Proteínas Munc18/metabolismo , Nanopartículas/química , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Animais , Área Sob a Curva , Toxinas Botulínicas/metabolismo , Humanos , Modelos Moleculares , Células PC12 , Domínios Proteicos , Estrutura Secundária de Proteína , RatosRESUMO
Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.
Assuntos
Endocitose , Movimento (Física) , Fenômenos Ópticos , Vesículas Sinápticas/metabolismo , Animais , Axônios/metabolismo , Teorema de Bayes , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Imageamento Tridimensional , Cadeias de Markov , Neurônios/metabolismo , Ratos Sprague-Dawley , Anticorpos de Domínio Único/metabolismo , Processos Estocásticos , Sinapses/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
In neurosecretory cells, secretory vesicles (SVs) undergo Ca(2+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking.
Assuntos
Actinas/metabolismo , Células Cromafins/fisiologia , Proteínas Munc18/metabolismo , Miosina Tipo II/metabolismo , Neurossecreção , Vesículas Secretórias/fisiologia , Animais , Bovinos , Compostos Heterocíclicos de 4 ou mais Anéis , Células PC12 , RatosRESUMO
How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.
Assuntos
Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Transporte Biológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Citocalasina D/farmacologia , Cinética , Microscopia Confocal , Microtúbulos/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Multimerização Proteica , Tiazolidinas/farmacologia , Imagem com Lapso de Tempo , Moduladores de Tubulina/farmacologiaRESUMO
Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.
Assuntos
Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutação Puntual , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Exocitose/fisiologia , Humanos , Modelos Moleculares , Proteínas Munc18/química , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Proteínas SNARE/metabolismo , UbiquitinaçãoRESUMO
Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma, and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca(2+) to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones. Here we discuss the functions of the cortical actin network, myosins, and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.
RESUMO
Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane.
Assuntos
Actinas/metabolismo , Exocitose , Cadeias Pesadas de Miosina/metabolismo , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Bovinos , Técnicas de Silenciamento de Genes , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Peptídeos/química , Fosforilação , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratos , Vesículas Secretórias/ultraestrutura , Quinases da Família src/metabolismoRESUMO
The sorting and concentration of cargo proteins within ER exit sites (ERESs) is a fundamental function of the secretory machinery. The mechanism by which peripheral coat complexes and their small GTPase effectors mediate this function with export membrane domains is only partially understood. The secretory-machinery-mediated sorting to ERESs is a process that counters the entropy-driven even distribution of membrane proteins within organellar membranes. Here, for the first time, we quantified the dynamic properties of GFP-VSVG sorting to ERESs in living cells by uncoupling it from later translocation steps using microtubule depolymerization. The dynamics of the ER to ERES redistribution of cargo proteins was quantified in single cells by measuring changes in fluorescence-intensity variance after shift to the permissive temperature. Cargo concentration within ERESs continued in cells overexpressing the GTP-locked ARF1Q71L or in the presence of brefeldin A. In the absence of COPI and microtubules, ERESs transformed from tubulovesicular to spherical membranes that actively accumulated secretory cargo and excluded ER-membrane markers. We found sorting to ERESs to be a slow and diffusion-unlimited process. Our findings exclude COPI, and identify the COPII protein complex to be directly involved in the secretory cargo sorting and redistribution functions of ERESs.