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1.
Cancer Res ; 81(8): 2234-2245, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33622696

RESUMO

Targeted imaging and therapy approaches based on novel prostate-specific membrane antigen (PSMA) inhibitors have fundamentally changed the treatment regimen of prostate cancer. However, the exact mechanism of PSMA inhibitor internalization has not yet been studied, and the inhibitors' subcellular fate remains elusive. Here, we investigated the intracellular distribution of peptidomimetic PSMA inhibitors and of PSMA itself by stimulated emission depletion (STED) nanoscopy, applying a novel nonstandard live cell staining protocol. Imaging analysis confirmed PSMA cluster formation at the cell surface of prostate cancer cells and clathrin-dependent endocytosis of PSMA inhibitors. Following the endosomal pathway, PSMA inhibitors accumulated in prostate cancer cells at clinically relevant time points. In contrast with PSMA itself, PSMA inhibitors were found to eventually distribute homogeneously in the cytoplasm, a molecular condition that promises benefits for treatment as cytoplasmic and in particular perinuclear enrichment of the radionuclide carriers may better facilitate the radiation-mediated damage of cancerous cells. This study is the first to reveal the subcellular fate of PSMA/PSMA inhibitor complexes at the nanoscale and aims to inspire the development of new approaches in the field of prostate cancer research, diagnostics, and therapeutics. SIGNIFICANCE: This study uses STED fluorescence microscopy to reveal the subcellular fate of PSMA/PSMA inhibitor complexes near the molecular level, providing insights of great clinical interest and suggestive of advantageous targeted therapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2234/F1.large.jpg.


Assuntos
Citoplasma/metabolismo , Glutamato Carboxipeptidase II/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Neoplasias da Próstata/metabolismo , Animais , Antígenos de Superfície/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Peptidomiméticos/farmacocinética , Peptidomiméticos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Coloração e Rotulagem
2.
Mol Cell ; 78(2): 236-249.e7, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32101700

RESUMO

The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.


Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Fibroblastos , Camundongos
3.
Proc Natl Acad Sci U S A ; 115(34): E8047-E8056, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30082388

RESUMO

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.


Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Proteínas Luminescentes/metabolismo , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Sinapses/metabolismo , Córtex Visual , Animais , Proteína 4 Homóloga a Disks-Large/genética , Proteínas Luminescentes/genética , Camundongos , Sinapses/genética , Córtex Visual/citologia , Córtex Visual/metabolismo
4.
Proc Natl Acad Sci U S A ; 115(10): E2246-E2253, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29463719

RESUMO

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.


Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos , Centríolos/química , Centríolos/genética , Centrossomo/química , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Células HeLa , Humanos , Interfase , Microscopia , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica , Proteínas/química , Proteínas/genética , tRNA Metiltransferases
5.
Biomed Opt Express ; 8(3): 1390-1404, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28663836

RESUMO

The chemical basis for the alteration of the refractive properties of an intraocular lens with a femtosecond laser was investigated. Three different microscope setups have been used for the study: Laser Induced Fluorescence (LIF) microscopy, Raman microscopy and coherent anti-Stokes Raman Scattering (CARS) microscopy. Photo-induced hydrolysis of polymeric material in aqueous media produces two hydrophilic functional groups: acid group and alcohol group. The spectral signatures identify two of the hydrophilic polar molecules as N-phenyl-4-(phenylazo)-benzenamine (C18H15N3) and phenazine-1-carboxylic acid (C13H8N2O2). The change in hydrophilicity results in a negative refractive index change in the laser-treated areas.

6.
Chemistry ; 23(50): 12114-12119, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28370443

RESUMO

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

7.
ACS Nano ; 10(9): 8215-22, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27517329

RESUMO

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.


Assuntos
Tomografia com Microscopia Eletrônica , HIV-1 , Proteólise , Vírion , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Infecções por HIV , Humanos , Peptídeos
8.
Proc Natl Acad Sci U S A ; 113(13): 3442-6, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26984498

RESUMO

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.

9.
EMBO J ; 35(4): 389-401, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26783362

RESUMO

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.


Assuntos
Apoptose , Mitocôndrias/enzimologia , Membranas Mitocondriais/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismo , Citocromos c/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Membranas Mitocondriais/fisiologia , Permeabilidade
10.
Opt Express ; 23(24): 30891-903, 2015 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-26698722

RESUMO

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.


Assuntos
Aumento da Imagem/instrumentação , Lentes , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e Especificidade
11.
Nat Methods ; 12(9): 827-30, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26214129

RESUMO

Electro-optical scanning (>1,000 frames/s) with pixel dwell times on the order of the lifetime of the fluorescent molecular state renders stimulated emission depletion (STED) nanoscopy temporally stochastic. Photon detection from a molecule occurs stochastically in one of several scanning frames, and the spatial origin of the photon is known with subdiffraction precision. Images are built up by binning consecutive frames, making the time resolution freely adjustable. We demonstrated nanoscopy of vesicle motions in living Drosophila larvae and the cellular uptake of viral particles with 5- to 10-ms temporal resolution.


Assuntos
Aumento da Imagem/instrumentação , Sistemas Microeletromecânicos/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Nanotecnologia/instrumentação , Fotometria/instrumentação , Interpretação Estatística de Dados , Desenho de Equipamento , Análise de Falha de Equipamento , Processos Estocásticos
12.
PLoS One ; 10(5): e0124650, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25993380

RESUMO

In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/citologia , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Neurônios/citologia , 1-Propanol/química , Animais , Encéfalo/virologia , Linhagem Celular , Cricetinae , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/virologia , Vírus da Raiva , terc-Butil Álcool/química
13.
Nat Commun ; 6: 7127, 2015 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-25980788

RESUMO

The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution. Here we report the discovery that STED nanoscopy of several red-emitting commercially available quantum dots is in fact successfully realized by the increasingly popular 775 nm STED laser light. A resolution of presently ∼ 50 nm is demonstrated for single quantum dots, and sub-diffraction resolution is further shown for imaging of quantum-dot-labelled vimentin filaments in fibroblasts. The high quantum-dot photostability enables repeated STED recordings with >1,000 frames. In addition, we have evidence that the tendency of quantum-dot labels to blink is largely suppressed by combined action of excitation and STED beams. Quantum-dot STED significantly expands the realm of application of STED nanoscopy, and, given the high stability of these probes, holds promise for extended time-lapse imaging.

14.
Opt Express ; 23(1): 211-23, 2015 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-25835668

RESUMO

Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 "intensity minima"). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.

15.
Chemphyschem ; 15(11): 2331-6, 2014 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-24753024

RESUMO

Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.


Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Cor , Fluorescência
16.
PLoS One ; 8(4): e62893, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23658655

RESUMO

The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.


Assuntos
Córtex Auditivo/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/ultraestrutura , Animais , Córtex Auditivo/metabolismo , Fixadores , Corantes Fluorescentes , Expressão Gênica , Microscopia de Fluorescência/métodos , Microtomia , Fosforilação , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapsinas/genética , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismo
17.
Methods Mol Biol ; 950: 27-41, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23086868

RESUMO

Optical microscopy has become a key technology in the life sciences today. Its noninvasive nature provides access to the interior of intact and even living cells, where specific molecules can be precisely localized by fluorescent tagging. However, the attainable 3D resolution of an optical microscope has long been hampered by a comparatively poor resolution along the optic axis. By coherent focusing through two objective lenses, 4Pi microscopy improves the axial resolution by three- to fivefold. This primer is intended as a starting point for the design and operation of a 4Pi microscope of type A.


Assuntos
Microscopia/métodos , Fenômenos Ópticos , Estruturas do Núcleo Celular/metabolismo , Complexo de Golgi/metabolismo , Processamento de Imagem Assistida por Computador , Compostos de Sulfidrila/química
18.
Science ; 338(6106): 524-8, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-23112332

RESUMO

Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.


Assuntos
HIV-1/fisiologia , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , HIV-1/ultraestrutura , Humanos , Microscopia de Fluorescência , Nanotecnologia/métodos , Multimerização Proteica , Proteólise
19.
J Neurosci ; 32(16): 5398-413, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22514304

RESUMO

BDNF plays a critical role in the regulation of synaptic strength and is essential for long-term potentiation, a phenomenon that underlies learning and memory. However, whether BDNF acts in a diffuse manner or is targeted to specific neuronal subcompartments or synaptic sites to affect circuit function remains unknown. Here, using photoactivation of BDNF or syt-IV (a regulator of exocytosis present on BDNF-containing vesicles) in transfected rat hippocampal neurons, we discovered that distinct subsets of BDNF vesicles are targeted to axons versus dendrites and are not shared between these compartments. Moreover, syt-IV- and BDNF-harboring vesicles are recruited to both presynaptic and postsynaptic sites in response to increased neuronal activity. Finally, using syt-IV knockout mouse neurons, we found that syt-IV is necessary for both presynaptic and postsynaptic scaling of synaptic strength in response to changes in network activity. These findings demonstrate that BDNF-containing vesicles can be targeted to specific sites in neurons and suggest that syt-IV-regulated BDNF secretion is subject to spatial control to regulate synaptic function in a site-specific manner.


Assuntos
Axônios/metabolismo , Dendritos/metabolismo , Neurônios/citologia , Vesículas Sinápticas/classificação , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Molécula de Adesão de Leucócito Ativado/metabolismo , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Técnicas de Cocultura , Colforsina/farmacologia , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Glicina/farmacologia , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Receptores de AMPA/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/fisiologia , Sinaptofisina/metabolismo , Sinaptotagminas/deficiência , Tetrodotoxina/farmacologia , Fatores de Tempo , Transfecção , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
20.
Opt Express ; 19(24): 23716-26, 2011 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-22109398

RESUMO

We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m.


Assuntos
Tecnologia de Fibra Óptica/instrumentação , Aumento da Imagem/instrumentação , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Refratometria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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