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1.
EMBO J ; 39(16): e104596, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32627850

RESUMO

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology.


Assuntos
Hipocampo/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Hipocampo/citologia , Neurônios/citologia , Transporte Proteico , Ratos
2.
EMBO J ; 37(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950309

RESUMO

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non-recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24-48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.


Assuntos
Hipocampo/citologia , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Espectrometria de Massas , Biossíntese de Proteínas/fisiologia , Ratos
3.
EMBO Rep ; 19(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29987134

RESUMO

Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high-quality super-resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.


Assuntos
Microscopia de Fluorescência/métodos , Acrilamida/química , Animais , Linhagem Celular , Cerebelo/ultraestrutura , Chlorocebus aethiops , Etilenodiaminas/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Neurônios/ultraestrutura , Peroxissomos/ultraestrutura , Polimerização , Compostos de Potássio/química , Ratos , Ratos Wistar , Sulfatos/química , Sinapses/ultraestrutura , Tubulina (Proteína)/ultraestrutura
5.
Methods Cell Biol ; 161: 33-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33478696

RESUMO

Expansion microscopy is a recently developed super-resolution imaging technique, which provides an alternative to optics-based methods such as deterministic approaches (e.g. STED) or stochastic approaches (e.g. PALM/STORM). The idea behind expansion microscopy is to embed the biological sample in a swellable gel, and then to expand it isotropically, thereby increasing the distance between the fluorophores. This approach breaks the diffraction barrier by simply separating the emission point-spread-functions of the fluorophores. The resolution attainable in expansion microscopy is thus directly dependent on the separation that can be achieved, i.e. on the expansion factor. The original implementation of the technique achieved an expansion factor of fourfold, for a resolution of 70-80nm. The subsequently developed X10 method achieves an expansion factor of 10-fold, for a resolution of 25-30nm. This technique can be implemented with minimal technical requirements on any standard fluorescence microscope, and is more easily applied for multi-color imaging than either deterministic or stochastic super-resolution approaches. This renders X10 expansion microscopy a highly promising tool for new biological discoveries, as discussed here, and as demonstrated by several recent applications.


Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência
6.
Cell Rep ; 34(11): 108841, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33730575

RESUMO

Synaptic transmission relies on the continual exocytosis and recycling of synaptic vesicles. Aged vesicle proteins are prevented from recycling and are eventually degraded. This implies that active synapses would lose vesicles and vesicle-associated proteins over time, unless the supply correlates to activity, to balance the losses. To test this hypothesis, we first model the quantitative relation between presynaptic spike rate and vesicle turnover. The model predicts that the vesicle supply needs to increase with the spike rate. To follow up this prediction, we measure protein turnover in individual synapses of cultured hippocampal neurons by combining nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorescence microscopy. We find that turnover correlates with activity at the single-synapse level, but not with other parameters such as the abundance of synaptic vesicles or postsynaptic density proteins. We therefore suggest that the supply of newly synthesized proteins to synapses is closely connected to synaptic activity.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Feminino , Fluorescência , Homeostase , Proteínas de Arcabouço Homer/metabolismo , Masculino , Modelos Neurológicos , Nanotecnologia , Biossíntese de Proteínas , Ratos Wistar , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo
7.
Nat Protoc ; 14(3): 832-863, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778205

RESUMO

Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60-80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.


Assuntos
Microscopia/métodos , Animais , Técnicas de Cultura de Células , Drosophila , Processamento de Imagem Assistida por Computador , Controle de Qualidade , Ratos Wistar , Peixe-Zebra
8.
Front Cell Neurosci ; 8: 409, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25538561

RESUMO

The trigger for synaptic vesicle exocytosis is Ca(2+), which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca(2+) levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca(2+) sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca(2+). The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca(2+) fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

9.
J Cell Biol ; 205(4): 591-606, 2014 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-24862576

RESUMO

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.


Assuntos
Membrana Celular/fisiologia , Microscopia de Fluorescência/métodos , Organelas/fisiologia , Transporte Proteico/fisiologia , Animais , Bactérias , Células COS , Chlorocebus aethiops , Drosophila melanogaster , Endocitose/fisiologia , Exocitose/fisiologia , Corantes Fluorescentes , Células Ciliadas Auditivas Internas/fisiologia , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Órgão Espiral/fisiologia , Cultura Primária de Células , Ratos , Saccharomyces cerevisiae , Vesículas Sinápticas/fisiologia
10.
Science ; 344(6187): 1023-8, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24876496

RESUMO

Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an "average" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.


Assuntos
Encéfalo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Encéfalo/ultraestrutura , Exocitose , Imageamento Tridimensional , Immunoblotting/métodos , Espectrometria de Massas/métodos , Microscopia Eletrônica/métodos , Modelos Neurológicos , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico , Ratos , Ratos Wistar , Vesículas Sinápticas/química , Sinaptossomos/química , Sinaptossomos/ultraestrutura , Proteínas de Transporte Vesicular/análise
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