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1.
Chemistry ; 27(14): 4670-4675, 2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33368712

RESUMO

Cytosolic protein delivery remains elusive. The inability of most proteins to cross the cellular membrane is a huge hurdle. Here we explore the unique photothermal properties of gold nanorods (AuNRs) to trigger cytosolic delivery of proteins. Both partners, protein and AuNRs, are modified with a protease-resistant cell-penetrating peptide with nuclear targeting properties to induce internalization. Once internalized, spatiotemporal control of protein release is achieved by near-infrared laser irradiation in the safe second biological window. Importantly, catalytic amounts of AuNRs are sufficient to trigger cytosolic protein delivery. To the best of our knowledge, this is the first time that AuNRs with their maximum of absorption in the second biological window are used to deliver proteins into the intracellular space. This strategy represents a powerful tool for the cytosolic delivery of virtually any class of protein.


Assuntos
Nanopartículas Metálicas , Nanotubos , Linhagem Celular Tumoral , Ouro , Fototerapia
2.
Nat Mater ; 18(4): 397-405, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30778227

RESUMO

The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.


Assuntos
Espaço Extracelular/metabolismo , Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Tecidos/métodos , Diferenciação Celular , Microambiente Celular , Feminino , Humanos , Cinética , Células-Tronco Pluripotentes/metabolismo , Gravidez , Terceiro Trimestre da Gravidez , Transcriptoma
3.
Nat Mater ; 14(3): 343-51, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25664452

RESUMO

The origin of fracture in epithelial cell sheets subject to stretch is commonly attributed to excess tension in the cells' cytoskeleton, in the plasma membrane, or in cell-cell contacts. Here, we demonstrate that for a variety of synthetic and physiological hydrogel substrates the formation of epithelial cracks is caused by tissue stretching independently of epithelial tension. We show that the origin of the cracks is hydraulic; they result from a transient pressure build-up in the substrate during stretch and compression manoeuvres. After pressure equilibration, cracks heal readily through actomyosin-dependent mechanisms. The observed phenomenology is captured by the theory of poroelasticity, which predicts the size and healing dynamics of epithelial cracks as a function of the stiffness, geometry and composition of the hydrogel substrate. Our findings demonstrate that epithelial integrity is determined in a tension-independent manner by the coupling between tissue stretching and matrix hydraulics.


Assuntos
Células Epiteliais/química , Células Epiteliais/fisiologia , Mecanotransdução Celular/fisiologia , Micromanipulação , Modelos Biológicos , Animais , Cães , Hidrodinâmica , Células Madin Darby de Rim Canino , Pressão , Estresse Mecânico , Propriedades de Superfície , Resistência à Tração/fisiologia
4.
bioRxiv ; 2023 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-37986921

RESUMO

The cell nucleus is continuously exposed to external signals, of both chemical and mechanical nature. To ensure proper cellular response, cells need to regulate not only the transmission of these signals, but also their timing and duration. Such timescale regulation is well described for fluctuating chemical signals, but if and how it applies to mechanical signals reaching the nucleus is still unknown. Here we demonstrate that the formation of fibrillar adhesions locks the nucleus in a mechanically deformed conformation, setting the mechanical response timescale to that of fibrillar adhesion remodelling (~1 hour). This process encompasses both mechanical deformation and associated mechanotransduction (such as via YAP), in response to both increased and decreased mechanical stimulation. The underlying mechanism is the anchoring of the vimentin cytoskeleton to fibrillar adhesions and the extracellular matrix through plectin 1f, which maintains nuclear deformation. Our results reveal a mechanism to regulate the timescale of mechanical adaptation, effectively setting a low pass filter to mechanotransduction.

5.
Nat Cell Biol ; 25(1): 120-133, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36543981

RESUMO

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.


Assuntos
Cavéolas , Caveolina 1 , Cavéolas/metabolismo , Caveolina 1/metabolismo , Mecanotransdução Celular , Membrana Celular/metabolismo , Proteínas/metabolismo
6.
Nat Commun ; 12(1): 6550, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34772909

RESUMO

In many physiological situations, BAR proteins reshape membranes with pre-existing curvature (templates), contributing to essential cellular processes. However, the mechanism and the biological implications of this reshaping process remain unclear. Here we show, both experimentally and through modelling, that BAR proteins reshape low curvature membrane templates through a mechanochemical phase transition. This phenomenon depends on initial template shape and involves the co-existence and progressive transition between distinct local states in terms of molecular organization (protein arrangement and density) and membrane shape (template size and spherical versus cylindrical curvature). Further, we demonstrate in cells that this phenomenon enables a mechanotransduction mode, in which cellular stretch leads to the mechanical formation of membrane templates, which are then reshaped into tubules by BAR proteins. Our results demonstrate the interplay between membrane mechanics and BAR protein molecular organization, integrating curvature sensing and generation in a comprehensive framework with implications for cell mechanical responses.


Assuntos
Mecanotransdução Celular/fisiologia , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Biologia Computacional , Humanos , Bicamadas Lipídicas/química , Mecanotransdução Celular/genética , Proteínas de Membrana/genética , Microscopia de Fluorescência
7.
Opt Express ; 16(17): 13315-22, 2008 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-18711568

RESUMO

We report for the first time the use of two photon fluorescence as detection method of affinity binding reactions. We use a resonant grating waveguide structure as platform enhancement for detecting the interaction between fluorescent labeled Boldenone, a non-natural androgenic hormone, and a specific anti-anabolic antibody. We were able to detect a surface coverage of approximately 0.7 ng/mm(2).


Assuntos
Androgênios/análise , Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Imunoensaio/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Fotometria/instrumentação , Refratometria/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Dinâmica não Linear , Transdutores
8.
Opt Express ; 14(14): 6394-9, 2006 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-19516817

RESUMO

In this paper, we describe the properties of Fabry-Perot fiber cavity formed by two fiber Bragg gratings in terms of the grating effective length. We show that the grating effective length is determined by the group delay of the grating, which depends on its diffraction efficiency and physical length. We present a simple analytical formula for calculation of the effective length of the uniform fiber Bragg grating and the frequency separation between consecutive resonances of a Fabry-Perot cavity. Experimental results on the cavity transmission spectra for different values of the gratings' reflectivity support the presented theory.

9.
Light Sci Appl ; 5(6): e16084, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30167169

RESUMO

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

10.
Biomed Opt Express ; 2(2): 305-14, 2011 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-21339876

RESUMO

Electroporation of neurons, i.e. electric-field induced generation of membrane nanopores to facilitate internalization of molecules, is a classic technique used in basic neuroscience research and recently has been proposed as a promising therapeutic strategy in the area of neuro-oncology. To optimize electroporation parameters, optical techniques capable of delivering time and spatially-resolved information on electroporation pore formation at the nanometer scale would be advantageous. For this purpose we describe here a novel optical method based on second harmonic generation (SHG) microscopy. Due to the nonlinear and coherent nature of SHG, the 3D radiation lobes from stained neuronal membranes are sensitive to the spatial distribution of scatterers in the illuminated patch, and in particular to nanopore formation.We used phase-array analysis to computationally study the SHG signal as a function of nanopore size and nanopore population density and confirmed experimentally, in accordance with previous work, the dependence of nanopore properties on membrane location with respect to the electroporation electric field; higher nanopore densities, lasting < 5 milliseconds, are observed at membrane patches perpendicular to the field whereas lower density is observed at partly tangent locations. Differences between near-anode and near-cathode cell poles are also measured, showing higher pore densities at the anodic pole compared to cathodic pole. This technique is promising for the study of nanopore dynamics in neurons and for the optimization of novel electroporation-based therapeutic approaches.

11.
Rev Sci Instrum ; 80(7): 073701, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19655950

RESUMO

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.


Assuntos
Microscopia Confocal/instrumentação , Nanotecnologia/instrumentação , Dinâmica não Linear , Animais , Caenorhabditis elegans , Desenho de Equipamento , Luz , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microcirurgia/instrumentação , Software
12.
J Opt Soc Am A Opt Image Sci Vis ; 20(10): 1981-6, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14570113

RESUMO

The field amplitude associated with ultrashort light pulses was analyzed by using the phase-space formalism of the Wigner distribution function (WDF). The diffraction integral was properly modified to take into account the dispersion effects (up to second order). A two-dimensional WDF associated with a reduced pupil function was derived, from which the on-axis irradiance was obtained for varying times. A two-dimensional and rotationally symmetric quartic-phase mask to control the temporal stretching of femtosecond light pulses passing through optical systems was proposed and analyzed. A Gaussian spatial and temporal pulse passing through a single lens with and without the phase mask was investigated.

13.
Appl Opt ; 43(15): 3005-9, 2004 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15176186

RESUMO

The analogy between free-space propagation of optical beams and light-pulse reflection from linearly chirped fiber gratings is used to analyze the Lau effect in the temporal domain. The coherence conditions that are satisfied in the spatial domain for obtaining, at certain fixed locations, periodic fringes patterns are reformulated for guided light propagation. In this analogy, spatial periodic irradiance distributions are transformed in periodic sequences of light pulses. An optical setup is proposed to produce sharp pulse trains, with minimal distortion effects, that have repetition frequencies that are different from those associated with the input periodic optical signal. Some numerical results are given to illustrate this approach.

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