Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
Cell ; 184(9): 2412-2429.e16, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33852913

RESUMO

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Biológico Ativo , Células HeLa , Humanos , Transporte Proteico
2.
Nature ; 626(7997): 169-176, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38267577

RESUMO

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.


Assuntos
Retículo Endoplasmático , Mitocôndrias , Membranas Mitocondriais , Movimento , Proteínas de Transporte Vesicular , Humanos , Esclerose Lateral Amiotrófica/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/ultraestrutura , Microscopia Eletrônica , Imageamento Tridimensional , Sítios de Ligação , Difusão , Fatores de Tempo , Mutação , Homeostase
4.
J Phys Chem A ; 122(14): 3594-3605, 2018 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-29558802

RESUMO

The time-resolved femtosecond stimulated Raman spectra (FSRS) of a charge transfer (CT) excited noncovalent complex tetracyanoethylene:1-chloronaphthalene (TCNE:ClN) in dichloromethane (DCM) is reported with 40 fs time resolution. In the frequency domain, five FSRS peaks are observed with frequencies of 534, 858, 1069, 1392, and 1926 cm-1. The most intense peaks at 534 and 1392 cm-1 correspond to fundamentals while the features at 858, 1069, and 1926 cm-1 are attributed to a difference frequency, an overtone and a combination frequency of the fundamentals, respectively. The frequency of the 1392 cm-1 fundamental corresponding to the central C═C stretch of TCNE•- is red-shifted from the frequency of the steady state radical due to the close proximity and electron affinity of the countercation. The observation of a FSRS band at a difference frequency is analyzed. This analysis lends evidence for alternative nonlinear pathways of inverse Raman gain scattering (IRGS) or vertical-FSRS (VFSRS) which may contribute to the time-evolving FSRS spectrum on-resonance. Impulsive stimulated Raman measurements of the complex show coherent oscillations of the stimulated emission with frequencies of 153, 278, and 534 cm-1. The 278 cm-1 mode corresponds to Cl bending of the dichloromethane solvent. The center frequency of the 278 cm-1 mode is modulated by a frequency of ∼30 cm-1 which is attributed to the effect of librational motion of the dichloromethane solvent as it reorganizes around the nascent contact ion pair. The 153 ± 15 cm-1 mode corresponds to an out-of-plane bending motion of TCNE. This motion modulates the intermolecular separation of the contact ion pair and thereby the overlap of the frontier orbitals which is crucial for rapid charge recombination in 5.9 ± 0.2 ps. High time-frequency resolution vibrational spectra provide unique molecular details regarding charge localization and recombination.

5.
Acc Chem Res ; 49(4): 616-25, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27003235

RESUMO

Femtosecond spectroscopy has revealed coherent wave packet motion time and time again, but the question as to whether these coherences are necessary for reactivity or merely a consequence of the experiment has remained open. For diatomic systems in the gas phase, such as sodium iodide, the dimensionality of the system requires coordinated atomic motion along the reaction coordinate. Coherent dynamics are also readily observed in condensed-phase multidimensional systems such as chromophores in proteins and solvated charge transfer dimers. Is precisely choreographed nuclear motion (i.e., coherence) required for reactivity in these systems? Can this coherence reveal anything about the reaction coordinate? In this Account, we describe our efforts to tackle these questions using femtosecond stimulated Raman spectroscopy (FSRS). Results of four exemplary systems are summarized to illustrate the role coherence can play in condensed-phase reactivity, the exploitation of vibrational coherence to measure vibrational anharmonicities, and the development of two-dimensional FSRS (2D-FSRS). We begin with rhodopsin, the protein responsible for vertebrate vision. The rhodopsin photoreaction is preternaturally fast: ground-state photoproduct is formed in less than 200 fs. However, the reactively important hydrogen out-of-plane motions as well as various torsions and stretches remain vibrationally coherent long after the reaction is complete, indicating that vibrational coherence can and does survive reactive internal conversion. Both the ultrashort time scale of the reaction and the observed vibrational coherence indicate that the reaction in rhodopsin is a vibrationally coherent process. Next we examine the functional excited-state proton transfer (ESPT) reaction of green fluorescent protein. Oscillations in the phenoxy C-O and imidazolinone C═N stretches in the FSRS spectrum indicated strong anharmonic coupling to a low-frequency phenyl wagging mode that gates the ESPT reaction. In this case, the coherence revealed not only itself but also the mode coupling that is necessary for reactivity. Curious as to whether vibrational coherence is a common phenomenon, we examined two simpler photochemical systems. FSRS studies of the charge transfer dimer tetramethylbenzene:tetracyanoquinodimethane revealed many vibrational oscillations with high signal-to-noise ratio that allowed us to develop a 2D-FSRS technique to quantitatively measure anharmonic vibrational coupling for many modes within a reacting excited state. Armed with this technique, we turned our attention to a bond-breaking reaction, the cycloreversion of a cyclohexadiene derivative. By means of 2D-FSRS, the vibrational composition of the excited-state transition state and therefore the reaction coordinate was revealed. In aggregate, these FSRS measurements demonstrate that vibrational coherences persist for many picoseconds in condensed phases at room temperature and can survive reactive internal conversion. Moreover, these coherences can be leveraged to reveal quantitative anharmonic couplings between a molecule's normal modes in the excited state. These anharmonic couplings are the key to determining how normal modes combine to form a reaction coordinate. It is becoming clear that condensed-phase photochemical reactions that occur in 10 ps or less require coordinated, coherent nuclear motion for efficient reactive internal conversion.

6.
Phys Chem Chem Phys ; 17(14): 9231-40, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25761064

RESUMO

Two-dimensional femtosecond stimulated Raman spectroscopy (2D-FSRS) is used to probe the structural evolution of a modified cyclohexadiene as it undergoes a photoinduced ring opening reaction. Analysis of the excited state stimulated Raman vibrational data reveals oscillations of the center frequencies and amplitudes of 21 high frequency modes. These oscillations in vibrational properties are due to anharmonic couplings between the high frequency finger print modes and the impulsively driven low frequency molecular distortions in the excited state. The largest anharmonic couplings, with intrinsic oscillation magnitudes of up to 40 cm(-1), are observed between the 467 cm(-1) C-C bend and the 1333 cm(-1) C-C stretch with the 191 cm(-1) methyl wag, all of which are centered on the reactive cyclohexadiene moiety. Conversely, motions located on the periphery - the 993 cm(-1) phenyl bend, the 1389 cm(-1) methyl bend and 1580 cm(-1) phenyl C-C stretch - are coupled with the 104 cm(-1) asymmetric bend. These couplings reveal two key energetic pathways: one leading to formation of the ring-opened product and the other reversion back to the ground state. This work is also important because it presents a new powerful method for measuring anharmonicities of potential energy surfaces and determining their role in chemical reactivity.

7.
J Phys Chem A ; 118(27): 4955-65, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-24932925

RESUMO

Photochemical reactions are mediated by conical intersections (CI), which are difficult to directly probe and characterize. To gain insight into CIs, two-dimensional femtosecond stimulated Raman spectroscopy (2D-FSRS) is used to examine a model excited-state charge-transfer (CT) complex consisting of an electron donor, tetramethylbenzene (TMB), and an acceptor, tetracyanoquinodimethane (TCNQ). Following impulsive excitation, the excited-state transient absorption reveals large-amplitude excited-state wave packet motion along low-frequency modes, in particular TCNQ's totally symmetric 323 cm(-1) CCN bend, which persist for ∼5 ps. These low-frequency coherences modulate the intensity and peak frequencies of the excited-state FSRS vibrational spectra. In particular, large-magnitude oscillations at 323 cm(-1) are observed in the peak frequency (Δω = 2 cm(-1)) and intensity (ΔOD = 1.5 mOD) of the nontotally symmetric 1271 cm(-1) C═C rocking mode. The magnitude of these oscillations is analyzed to determine the first-order anharmonic coupling between the high- and low-frequency degrees of freedom in the excited state. The anharmonic coupling between the totally symmetric 323 cm(-1) and the nontotally symmetric 1271 cm(-1) modes is estimated to be in excess of 50 cm(-1), strongly suggesting that they are the tuning and coupling modes, respectively, for the CI that connects the CT excited state to the neutral ground state and controls charge recombination internal conversion.

8.
Opt Express ; 21(18): 21685-92, 2013 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-24104042

RESUMO

A comparison between a Fabry-Pérot etalon filter and a conventional grating filter for producing the picosecond (ps) Raman pump pulses for femtosecond stimulated Raman spectroscopy (FSRS) is presented. It is shown that for pulses of equal energy the etalon filter produces Raman signals twice as large as that of the grating filter while suppressing the electronically resonant background signal. The time asymmetric profile of the etalon-generated pulse is shown to be responsible for both of these observations. A theoretical discussion is presented which quantitatively supports this hypothesis. It is concluded that etalons are the ideal method for the generation of narrowband ps pulses for FSRS because of the optical simplicity, efficiency, improved FSRS intensity and reduced backgrounds.

9.
J Phys Chem A ; 117(45): 11472-8, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24138073

RESUMO

Azobenzenes are versatile photoswitches that find application in optical memory, light-driven motors, and molecular gating. Despite many studies, the molecular details of their light induced trans to cis isomerization are still debated. To inform this discussion we probed the low frequency skeletal motions in an azobenzene derivative, 4-nitro-4'-dimethylamino-azobenzene (NDAB), with resonant impulsive stimulated Raman spectroscopy (RISRS). Four previously unobserved modes at 14, 47, 150, and 201 cm(-1) were found. Of these, the ∼50 cm(-1) inversion motion and the ∼15 cm(-1) torsional motion had particularly large intensities, suggesting that the excited state potential energy surface is steeply sloped along these coordinates in the Franck-Condon region. These data support a model in which NDAB isomerizes predominantly along a prompt inversion coordinate as well as a slower torsional motion that mitigates the phenyl-phenyl interactions on the pathway to the isomerized product.

10.
Phys Chem Chem Phys ; 14(18): 6298-306, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22370582

RESUMO

Azobenzenes are used in many applications because of their robust and reversible light induced trans⇋cis isomerization about the N=N bond, but the mechanism of this ultrafast reaction has not been conclusively defined. Addressing this problem we have used Femtosecond Stimulated Raman Spectroscopy (FSRS) to determine the structural transients in the trans→cis photoisomerization of the azobenzene derivative, 4-nitro-4'-dimethylamino-azobenzene (NDAB). Key marker modes, such as the 1570/1590 cm(-1) NO(2) stretch and the 1630 cm(-1) C-N(Me)(2) stretch, enable the separation and analysis of distinct trans and cis photoproduct dynamics revealing the 400 fs Frank-Condon relaxation, the 800 fs timescale of the cis product formation and the 2 ps emergence and 8 ps relaxation of the unsuccessful ground state trans species. Based on these observations, we propose a reaction mechanism, including initial dilation of the CNN bend later joined by quick movement along the CCNN, CNNC and NNCC torsional coordinates that constitutes a mixed inversion-rotation mechanism.


Assuntos
Compostos de Anilina/química , Compostos Azo/química , Processos Fotoquímicos , Análise Espectral Raman , Fatores de Tempo , Vibração
12.
Science ; 376(6591): 377-382, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35446649

RESUMO

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Glicoproteínas de Membrana , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Granzimas/metabolismo , Glicoproteínas de Membrana/metabolismo , Perforina/genética , Perforina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Linfócitos T Citotóxicos/metabolismo
13.
Science ; 367(6475)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31949053

RESUMO

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.


Assuntos
Células/ultraestrutura , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Células COS , Adesão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Congelamento , Células HeLa , Humanos , Camundongos
14.
J Phys Chem Lett ; 6(15): 2919-23, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26267181

RESUMO

Exciton mobility is crucial to organic photovoltaic (OPV) efficiency, but accurate, quantitative measures and therefore precise understanding of this process are currently lacking. Here, we exploit the unique capabilities of femtosecond stimulated Raman spectroscopy (FSRS) to disentangle the signatures of the bulk and interfacial donor response in a bulk heterojunction composed of poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1,4-phenylenevinylene] (MDMO-PPV) and phenyl-C61-butyric acid methyl ester (PCBM). Surprisingly, we find that donor excitons are very mobile for the first ∼300 fs following excitation (before thermalization) even though their overall lifetime is significantly longer (170 ps). A sharp decrease in mobility occurs after the system relaxes out of the Franck-Condon (FC) region. From this observation we predict that any polymer lacking a significant resonance Raman effect and fluorescence Stokes shift, indicating slow FC relaxation and small reorganization energy, will make an efficient OPV material.

15.
J Public Health Manag Pract ; 11(1): 59-64, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15692294

RESUMO

From 1997 through 1999, a total of 365 diabetes screening and awareness events targeting high-risk populations were held throughout New York State. These events were planned and implemented by community-based coalitions that received funding from the state's Diabetes Control Program. The American Diabetes Association's diabetes risk questionnaire was administered, and those individuals identified as high risk received a capillary blood glucose test. Screened individuals with glucose readings above the cut-off value (140 mg/dl or 110 mg/dl if fasting) were referred to a physician for diagnostic testing. A total of 32,954 individuals took the questionnaire, 27,237 received the blood test, and 1,564 were referred to a physician. Among those who were successfully tracked (n = 1,113), 354 were newly diagnosed with diabetes mellitus. Seventy-two percent of participants screened were aged 45 years and older, and 67% had a body mass index of 25 or higher. Only 15% were members of ethnic minorities, and uninsured individuals were also underrepresented at 10%. The entire initiative, including planning, promotion, and administration, required 5,428 person-hours of staff time and a total cost of approximately 262,000 dollars. Fifty-seven percent of the total cost was derived from in-kind support of the coalitions. The cost of detecting each new case was 741 dollars.


Assuntos
Serviços de Saúde Comunitária/organização & administração , Diabetes Mellitus/diagnóstico , Programas de Rastreamento/economia , Avaliação de Resultados em Cuidados de Saúde , Administração em Saúde Pública , Marketing Social , Adulto , Idoso , Conscientização , Glicemia/análise , Custos e Análise de Custo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , New York , Avaliação de Programas e Projetos de Saúde , Medição de Risco , Inquéritos e Questionários
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA