Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Nature ; 562(7727): 361-366, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30333578

RESUMO

Few animals provide a readout that is as objective of their perceptual state as camouflaging cephalopods. Their skin display system includes an extensive array of pigment cells (chromatophores), each expandable by radial muscles controlled by motor neurons. If one could track the individual expansion states of the chromatophores, one would obtain a quantitative description-and potentially even a neural description by proxy-of the perceptual state of the animal in real time. Here we present the use of computational and analytical methods to achieve this in behaving animals, quantifying the states of tens of thousands of chromatophores at sixty frames per second, at single-cell resolution, and over weeks. We infer a statistical hierarchy of motor control, reveal an underlying low-dimensional structure to pattern dynamics and uncover rules that govern the development of skin patterns. This approach provides an objective description of complex perceptual behaviour, and a powerful means to uncover the organizational principles that underlie the function, dynamics and morphogenesis of neural systems.


Assuntos
Mimetismo Biológico/fisiologia , Cromatóforos/fisiologia , Decapodiformes/fisiologia , Fenômenos Fisiológicos da Pele , Animais , Comportamento Animal , Cor , Decapodiformes/citologia , Modelos Biológicos , Neurônios Motores/fisiologia , Análise de Célula Única , Pele/citologia
2.
EMBO J ; 37(1): 139-159, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29146773

RESUMO

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.


Assuntos
Fixadores/química , Formaldeído/química , Glioxal/química , Imuno-Histoquímica/métodos , Microscopia de Fluorescência/métodos , Proteínas do Tecido Nervoso/metabolismo , Fixação de Tecidos/métodos , Animais , Células COS , Chlorocebus aethiops , Drosophila melanogaster , Células HeLa , Humanos , Camundongos
3.
J Immunol ; 204(10): 2818-2828, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32269094

RESUMO

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H+)-adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.


Assuntos
Sinapses Imunológicas/metabolismo , Microtúbulos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Células Cultivadas , Citotoxicidade Imunológica , Exocitose , Concentração de Íons de Hidrogênio , Sinapses Imunológicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Proteínas R-SNARE/genética , Linfócitos T Citotóxicos/imunologia , ATPases Vacuolares Próton-Translocadoras/genética
4.
Int J Mol Sci ; 23(13)2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35806143

RESUMO

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.


Assuntos
Ciliopatias , Sinapses , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciliopatias/metabolismo , Proteínas Correpressoras/metabolismo , Humanos , Fosfoproteínas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo
5.
J Physiol ; 593(13): 2807-32, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25864578

RESUMO

KEY POINTS: Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca(2+) -independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. ABSTRACT: Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca(2+) -regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca(2+) -independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Cálcio/metabolismo , Vesículas Transportadoras/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Endocitose , Exocitose , Camundongos , Transporte Proteico
6.
Circ Res ; 111(4): 402-14, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22723297

RESUMO

RATIONALE: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca(2+) release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). OBJECTIVES: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. METHODS AND RESULTS: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca(2+) release and action potential prolongation. CONCLUSIONS: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca(2+) release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.


Assuntos
Membranas Intracelulares/patologia , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microtúbulos/patologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Nanotecnologia , Remodelação Ventricular , Potenciais de Ação , Animais , Caveolina 3/metabolismo , Simulação por Computador , Modelos Animais de Doenças , Acoplamento Excitação-Contração , Feminino , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Modelos Cardiovasculares , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de Tempo
7.
J Opt Soc Am A Opt Image Sci Vis ; 31(6): 1215-25, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24977359

RESUMO

We demonstrate quantitative phase delay measurements with a spiral phase contrast microscope working in confocal mode. Such a confocal configuration is sensitive to weak phase objects due to background rejection but does not give direct access to the phase delay introduced by the sample. We develop a theory showing that shifting the illumination spot relative to the detector gives access to the local phase gradient in the first-order approximation. Subsequently, we present an iterative integration algorithm for phase delay measurements. This approach is validated on simulated and calibrated experimental images. Finally, the algorithm is applied to measure the phase profile of a cell, in which phase delays of 10 mrad are observed.

8.
Proc Natl Acad Sci U S A ; 108(33): 13546-51, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21799113

RESUMO

The translocase of the mitochondrial outer membrane (TOM) complex is the main import pore for nuclear-encoded proteins into mitochondria, yet little is known about its spatial distribution within the outer membrane. Super-resolution stimulated emission depletion microscopy was used to determine quantitatively the nanoscale distribution of Tom20, a subunit of the TOM complex, in more than 1,000 cells. We demonstrate that Tom20 is located in clusters whose nanoscale distribution is finely adjusted to the cellular growth conditions as well as to the specific position of a cell within a microcolony. The density of the clusters correlates to the mitochondrial membrane potential. The distributions of clusters of Tom20 and of Tom22 follow an inner-cellular gradient from the perinuclear to the peripheral mitochondria. We conclude that the nanoscale distribution of the TOM complex is finely adjusted to the cellular conditions, resulting in distribution gradients both within single cells and between adjacent cells.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Fenômenos Fisiológicos Celulares , Proteínas de Membrana Transportadoras , Proteínas de Transporte da Membrana Mitocondrial/análise , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Ratos , Receptores de Superfície Celular , Receptores Citoplasmáticos e Nucleares/análise , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo
9.
Proc Natl Acad Sci U S A ; 107(44): 19055-60, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-20956291

RESUMO

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.


Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Exocitose/fisiologia , Modelos Biológicos , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Proteínas de Membrana/metabolismo , Camundongos , Células PC12 , Ratos
10.
Prog Neurobiol ; 231: 102541, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37898315

RESUMO

Dendritic spines are key structures for neural communication, learning and memory. Spine size and shape probably reflect synaptic strength and learning. Imaging with superresolution STED microscopy the detailed shape of the majority of the spines of individual neurons in turtle cortex (Trachemys scripta elegans) revealed several distinguishable shape classes. Dendritic spines of a given class were not distributed randomly, but rather decorated significantly more often some dendrites than others. The individuality of dendrites was corroborated by significant inter-dendrite differences in other parameters such as spine density and length. In addition, many spines were branched or possessed spinules. These findings may have implications for the role of individual dendrites in this cortex.


Assuntos
Dendritos , Tartarugas , Animais , Microscopia , Neurônios/fisiologia , Córtex Cerebral , Espinhas Dendríticas
11.
Front Mol Neurosci ; 15: 674243, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493323

RESUMO

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using - in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

12.
Biophys J ; 99(2): 675-84, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20643088

RESUMO

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.


Assuntos
Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Eletricidade , Endocitose , Fusão de Membrana , Microscopia , Ratos
13.
Langmuir ; 26(18): 14400-4, 2010 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-20715873

RESUMO

The dynamic noninvasive imaging of colloidal nanostructures has been precluded by the diffraction-limited resolution of (confocal) light microscopy. Using Fast Stimulated Emission Depletion (STED) microscopy, we demonstrate the ability to resolve the formation of a colloidal crystal (monolayer) from particles of 200 nm size, where the voids in the crystal are as small as 30 nm. With a temporal resolution of 5 ms, we exemplify the technique by visualizing the annealing of potential point defects during the formation of the colloidal crystal.


Assuntos
Coloides/química , Microscopia/métodos , Nanoestruturas/química , Fatores de Tempo
14.
J Clin Invest ; 129(5): 2145-2162, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30985297

RESUMO

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.


Assuntos
Sistema Nervoso Central/fisiopatologia , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/genética , Células-Tronco Pluripotentes/metabolismo , Receptores de Superfície Celular/genética , ATPases Vacuolares Próton-Translocadoras/genética , Adolescente , Processamento Alternativo , Animais , Apoptose , Encéfalo/diagnóstico por imagem , Morte Celular , Diferenciação Celular , Sobrevivência Celular , Pré-Escolar , Deleção de Genes , Variação Genética , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/fisiologia , Receptores de Superfície Celular/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia
15.
Opt Express ; 14(9): 3700-14, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19516517

RESUMO

We describe and analyze an interferometer-based virtual modal wavefront sensor (VMWS) that can be configured to measure, for example, Zernike coefficients directly. This sensor is particularly light efficient because the determination of each modal coefficient benefits from all the available photons. Numerical simulations show that the VMWS outperforms state-of-the-art phase unwrapping at low light levels. Including up to Zernike mode 21, aberrations can be determined with a precision of about 0.17 rad (lambda/37) using low resolution (65 x 65 pixels) images and only about 400 photons total.

16.
PLoS One ; 10(12): e0143681, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26625116

RESUMO

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.


Assuntos
Cálcio/metabolismo , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Neurônios Motores/metabolismo , Animais , Cálcio/análise , Embrião não Mamífero , Medula Espinal/embriologia , Medula Espinal/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
17.
Sci Rep ; 3: 2050, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23787399

RESUMO

Stimulated Emission Depletion (STED) microscopy enables superresolution imaging of fluorescently marked nano-structures in vivo. Biological investigations are often hindered by the difficulty of relating super-resolved structures to other non-labeled features. Here we demonstrate that the similarity in optical design of Spiral Phase Contrast (SPC) and STED microscopes allows straightforward implementation of a phase contrast channel into a STED microscope in widefield and scanning modes. This method allows dual imaging and overlay in two contrast modes in fixed and in living specimens, in which double labeling is especially challenging. Living GFP- and YPF-stained neurons are imaged in one label-free phase contrast and one high-resolution STED channel. Furthermore, we implement SPC in widefield and scanning modes demonstrating that scanning confocal SPC yields the highest optical contrast. The latter configuration can provide contour detection or highlights and shadows reminiscent of differential interference contrast.


Assuntos
Microscopia Confocal/instrumentação , Animais , Células Cultivadas , Camundongos , Microscopia Confocal/métodos , Neurônios/citologia , Neurônios/metabolismo
18.
Exp Hematol ; 41(9): 823-831.e2, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23660069

RESUMO

Homing and engraftment of hematopoietic stem and progenitor cells (HSPCs) during bone marrow transplantation are critically dependent on integrins such as ß1-integrin. In the present study, we show that ß1-integrin and the tetraspanin CD63 form a cell surface receptor complex for the soluble serum protein tissue inhibitor of metalloproteinases-1 (TIMP-1) on human CD34⁺ HSPCs. Through binding to this receptor complex, TIMP-1 activates ß1-integrin, increases adhesion and migration of human CD34⁺ cells, and protects these cells from induced apoptosis. TIMP-1 stimulation in murine bone marrow mononuclear cells also promotes migration and adhesion; this is associated with augmented homing of murine mononuclear cells and of murine LSK⁺ cells during bone marrow transplantation. These results not only indicate that TIMP-1 is conducive to HSPC homing; they also identify CD63 and ß1-integrin as a TIMP-1 receptor complex on HSPCs.


Assuntos
Transplante de Medula Óssea , Movimento Celular , Sobrevivência de Enxerto , Células-Tronco Hematopoéticas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Adesão Celular , Feminino , Humanos , Integrina beta1/metabolismo , Masculino , Camundongos , Tetraspanina 30/metabolismo , Transplante Homólogo
19.
J Biophotonics ; 3(7): 417-24, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20379984

RESUMO

We compare the performance of video-rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4-fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video-rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples.


Assuntos
Microscopia Confocal , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Neurônios/fisiologia , Gravação em Vídeo/métodos , Animais , Axônios/fisiologia , Células Cultivadas , Fluorescência , Hipocampo/citologia , Hipocampo/fisiologia , Processamento de Imagem Assistida por Computador , Movimento (Física) , Neurônios/citologia , Fótons , Ratos , Vesículas Sinápticas/fisiologia , Fatores de Tempo
20.
J Cell Biol ; 189(2): 223-32, 2010 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-20404108

RESUMO

Oligodendrocytes secrete vesicles into the extracellular space, where they might play a role in neuron-glia communication. These exosomes are small vesicles with a diameter of 50-100 nm that are formed within multivesicular bodies and are released after fusion with the plasma membrane. The intracellular pathways that generate exosomes are poorly defined. Because Rab family guanosine triphosphatases (GTPases) together with their regulators are important membrane trafficking organizers, we investigated which Rab GTPase-activating proteins interfere with exosome release. We find that TBC1D10A-C regulate exosome secretion in a catalytic activity-dependent manner. We show that Rab35 is the target of TBC1D10A-C and that the inhibition of Rab35 function leads to intracellular accumulation of endosomal vesicles and impairs exosome secretion. Rab35 localizes to the surface of oligodendroglia in a GTP-dependent manner, where it increases the density of vesicles, suggesting a function in docking or tethering. These findings provide a basis for understanding the biogenesis and function of exosomes in the central nervous system.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Exossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Oligodendroglia/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Exocitose/fisiologia , Proteínas Ativadoras de GTPase/genética , Humanos , Camundongos , Oligodendroglia/citologia , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA