Este estudo foi financiado pelo NIH o financiamento da investigação (DK090868 e GM092930 de TI). Existe uma patente pendente sobre um análogo rapamicina enjaulado.

1. A transfecção de DNA de plasmídeo <ol><li> Adicionar DNAs plasmídicos, 0,5 ug FRB membrana amarrado (daqui em diante referido como LDR) e 0,5 ug FKBP-Tiam1 (T-cell lymphoma invasão e metástase indutora de proteína 1: Rac activador) marcado com proteína fluorescente para 37,5 uL dH 2 O. Adicionar 1 ml FuGENE HD. </li><li> Vortex e incube à temperatura ambiente durante 20 minutos. Enquanto isso, vá para as etapas 1,3-1,8. </li><li> Lava-se cada poço de uma câmara de 8-bem com 50 uL de poli-D-lisina. </li><li> Lavar o centro de cada poço com dH 2 O. </li><li> Trypsinize 80-85% confluentes células NIH-3T3 e diluir até 10 mL meio de cultura (DMEM com FBS 10%). </li><li> Remover 375 ul de suspensão e centrifugar a 1750 rpm durante 3 minutos. </li><li> Aspirar a mídia, tomando cuidado para não perturbar o sedimento. </li><li> Adicionar 750 ul de DMEM FBS w / 10% e ressuspender. </li><li> Adicionar suspensão de células de dH 2 O / DNA / solução FuGENE HD e misture delicadamente. </li><li> Adicionar 250ul de célula / DNA suspensão a cada poço (irá preencher até 3 poços). </li><li> Incubar as células transfectadas a 37 ° C e 5% de CO2 durante 15-18 hr. </li></ol> 2. Preparação de crb-se uma solução <ol><li> Sintetizar enjaulado aduto rapamicina-biotina (CRB). Consulte &quot;esquema sintético do crb&quot; para detalhes. Resumidamente, porções enjaulamento e biotina são preparados separadamente. A porção de encarceramento é então conjugado de rapamicina (LC laboratório), o produto subsequentemente acoplado à porção de biotina por um meio de reacção clique 7 para se obter CRB. </li><li> Preparar uma solução-mãe 1 mM de crb em DMSO. </li><li> Dissolve-se avidina 1mg em 250 ul de PBS em um tubo Eppendorf 0,5 ml. </li><li> Adicionar 1 ml de solução estoque para crb avidina dissolvido. Misturar invertendo várias vezes. </li><li> Incubar a temperatura ambiente durante 15 min para se obter CRB-avidina conjugada (CRB-A) De solução. </li><li> Excluir pequenas moléculas não ligadas e purificar CRB-A. Utilizando uma coluna de exclusão de tamanho </li></ol> 3. Microscopia e Iluminação Luz em nível subcelular <ol><li> Soro fome células transfectadas em DMEM sem FBS durante 12 horas. </li><li> Aspirar cuidadosamente os meios de comunicação fora das células e adicionar 250 uL da nM 400 preparada CRB-se uma solução (a partir do passo 2.6). </li><li> Para visualizar dinâmica de proteínas em células vivas em resolução subcelular antes e após tratamento com luz, microscopia de disco giratório foi utilizado (Figura 2a). Iluminação UV foi conseguida por acoplamento óptica para luz do LED UV (365 nm) com epi-fluorescência de luz (Figura 2b). </li><li> MetaMorph software foi usado para capturar imagens de fluorescência de células transfectadas a cada 15 segundos. </li><li> Definir as coordenadas e tamanho de um ponto de iluminação usando uma placa de vidro revestida com corantes fluorescentes excitadas pela luz UV. O tamanho podeser variado através da aplicação de lente objectiva com ampliações diferentes e seleccionando as fibras ópticas com tamanho do núcleo diferente. </li><li> Após a determinação dos níveis de fundo de luz membrana ruffling UV, irradiam na periferia das células. Os dois passos seguintes são para contrastar anterior efeitos localizados. </li><li> Globalmente irradiar as células com luz de 365 nm fornecida por uma lâmpada de mercúrio. </li><li> Dissolver uncaged, assim rapamicina original em DMSO e em seguida em PBS (400 nM final), e adicionar a solução para a câmara de imagem para induzir efeitos globais. </li></ol> 4. Os resultados representativos Um exemplo de resultados representativos é mostrado na Figura 3. Irradiação com luz UV a uma pequena região perto de uma aresta celular rapidamente induzida a formação de zonas plissados ​​apenas na e próximo da área irradiada. Para confirmar que esta era de fato a atividade local, em seguida, globalmente irradiados células com UV, ou adicionado a rapamicina para os meios de comunicação em massa, tanto de which induziram a formação ruffle global ao longo da membrana. Para uma análise quantitativa, as células alvo dividida em quatro quadrantes, e contados a frequência de formação de plissado em cada quadrante sobre local, bem como após a estimulação global sob várias condições, incluindo os controlos (Figura 4). É apenas esta combinação de irradiação UV, CRB-A, e adequados FKBP-FRB construções que induz ruffling local. <img alt="A Figura 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> Figura 1. Esquemática da dimerização de proteínas espacialmente confinado usando gaiolas rapamicina-avidina conjugada (CRB-A) e luz UV. Irradiação UV cliva o ligante entre rapamicina e biotina, resultando na libertação de química dimerizer (HE-Rapa, C40-O-(2-hidroxietil) rapamicina) e do seu sub-produto. O dimerizer liberado em seguida, difunde para as células e induz a dimerização entre FKBP-POI (proteína de interesse) e memb plasmarane ancorado FRB apenas na proximidade da região irradiada (círculo azul). Sem a irradiação UV, FKBP e FRB não associado (cruz vermelha), portanto, não produzindo efeito. (Copyright @ ACS2011) 6 <img alt="A Figura 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> Figura 2. Imagem do microscópio confocal personalização de fluorescência. (A) Vista frontal do microscópio confocal (invertido Axiovert 200, Zeiss). (B) Close-up vista, topo de uma unidade de acoplamento de EPI fluorescência e iluminação UV. <img alt="A Figura 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> Figura 3. Formação plissado espacialmente confinado numa célula NIH3T3 transfectadas induzida por irradiação UV localizada. Confocal imagens de fluorescência de uma célula de fibroblastos NIH3T3 que é transfectada com YFP-marcado FKBP-Tiam1 (Rac ativador) e LDR (membrana ancoradas FRB) foram submetidos à irradiação em uma área restrita perto de sua borda com LED luz a 365 nm durante 15 seg 210 seg a partir de, na presença de CRB-Um conjugado nos meios de comunicação a granel. Este tratamento foi seguido de iluminação global com lâmpada de mercúrio durante 1 seg a 1063 adição seg e global de rapamicina finais 400 nM em 1560 seg. A célula mostrou quase nenhuma atividade despenteando no início (a), mas mostrou espacialmente confinado despenteando após a iluminação localizada (círculo amarelo) (b). A célula mostrou a formação plissado global após iluminação global e adição de rapamicina (cd), indicando que a formação de plissado local foi causada por activação espacialmente confinado de rac. Barra de escala: 10 um. (Copyright @ ACS2011) 6 <img alt="A Figura 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> Figura 4. Frequência de formação de plissado em quadrantes de transfectadas células NIH3T3. Irradiação UV local com CRB-A e irradiação UV seguinte global foram realizados em 13 células. CRB-A </sTrong&gt; adição (sem irradiação de UV), irradiação UV local com ou sem avidina foram aplicadas sobre as células 13, 5 e 6, respectivamente. Nenhuma das células observados mostrou a formação de plissado com CRB-A, sem irradiação de UV, ou irradiação de UV local com ou sem avidina em meios a granel. Foram colhidos dados a partir de células com baixa formação de fundo plissado, mas que tinha a capacidade de rapamicina induzida por formação de plissado, que verificada após cada experiência, confirmando a formação de plissado global como um resultado da adição de rapamicina (finais 400 nM). Estes resultados mostraram que CRB-A e irradiação UV local são necessários para a formação plissado local nas células. (Copyright @ ACS2011) 6 Abreviaturas: FKBP: FK506-binding protein FRB: FKBP-rapamicina-binding protein Tiam1: T-cell lymphoma invasão e metástase indutor de proteína 1 (Rac GEF) LDR: Lyn-DSAG ligante-FRB (Lyn: N-terminal 11 aminoácidos da Lyn quinase) HE-Rapa: C40-O - (2-hidroxietil) rapamicina crb: rapamicina enjaulado com porção biotina CRB-A: crb conjugada com avidina

<ol>
	<li>Etienne-Manneville, S., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+cell+biology.">Rho GTPases in cell biology.</a> Nature. 420, 629-635 (2002).</li><li>Takai, Y., Sasaki, T., Matozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTP-binding+proteins.">Small GTP-binding proteins.</a> Physiol. Rev. 81, 153-208 (2001).</li><li>Kiyokawa, E., Aoki, K., Nakamura, T., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+regulation+of+small+GTPases+as+revealed+by+probes+based+on+the+principle+of+Forster+Resonance+Energy+Transfer+(FRET):+Implications+for+signaling+and+pharmacology.">Spatiotemporal regulation of small GTPases as revealed by probes based on the principle of Forster Resonance Energy Transfer (FRET): Implications for signaling and pharmacology.</a> Annu. Rev. Pharmacol. Toxicol. 51, 337-358 (2011).</li><li>Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J., Meyer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+translocation+strategy+to+rapidly+activate+and+inhibit+small+GTPase+signaling+pathways.">An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways.</a> Nat. Methods. 2, 415-418 (2005).</li><li>Komatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific,+rapid+induction+of+molecular+activities+and+membrane+tethering.">Organelle-specific, rapid induction of molecular activities and membrane tethering.</a> Nat. Methods. 7, 206-208 (2010).</li><li>Umeda, N., Ueno, T., Pohlmeyer, C., Nagano, T., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photocleavable+rapamycin+conjugate+for+spatiotemporal+control+of+small+GTPase+activity.">A photocleavable rapamycin conjugate for spatiotemporal control of small GTPase activity.</a> J. Am .Chem. Soc. 133, 12-14 (2011).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry:+Diverse+Chemical+Function+from+a+Few+Good+Reactions.">Click Chemistry: Diverse Chemical Function from a Few Good Reactions.</a> Angew. Chem. Int. Ed. Engl. 40, 2004-2021 (2001).</li><li>Kennedy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nat. Methods. 7, 973-975 (2010).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461, 997-1001 (2009).</li><li>Wu, Y. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+photoactivatable+Rac+controls+the+motility+of+living+cells.">A genetically encoded photoactivatable Rac controls the motility of living cells.</a> Nature. 461, 104-108 (2009).</li><li>Yazawa, M., Sadaghiani, A. M., Hsueh, B., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+protein-protein+interactions+in+live+cells+using+light.">Induction of protein-protein interactions in live cells using light.</a> Nat. Biotechnol. 27, 941-945 (2009).</li><li>Fegan, A., White, B., Carlson, J. C., Wagner, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+protein+assembly:+techniques+and+applications.">Chemically controlled protein assembly: techniques and applications.</a> Chem. Rev. 110, 3315-3336 (2010).</li><li>Suh, B. C., Inoue, T., Meyer, T., Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+chemically+induced+changes+of+PtdIns(4,5)P2+gate+KCNQ+ion+channels.">Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.</a> Science. 314, 1454-1457 (2006).</li><li>Varnai, P., Thyagarajan, B., Rohacs, T., Balla, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapidly+inducible+changes+in+phosphatidylinositol+4,5-bisphosphate+levels+influence+multiple+regulatory+functions+of+the+lipid+in+intact+living+cells.">Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells.</a> J. Cell. Biol. 175, 377-382 (2006).</li></ol>

Não há conflitos de interesse declarados.

Nós descrevemos uma técnica que utiliza um novo composto engaiolados em conjunto com o sistema de heterodimerização FKBP-FRB, a fim de manipular Rho actividade GTPase a uma localização precisa subcelular numa escala de tempo de segundos. Há três limitações da presente abordagem. Em primeiro lugar, porque o método baseia-se na prevenção ou permitindo a difusão de dimerizer através da membrana plasmática, a localização do alvo celular necessita de ser membrana plasmática ou ...

<table border="1"><tbody><tr><td> Nome do reagente / equipamentos </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Rapamicina </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidina </td><td> Sigma </td><td> A9275-10MG </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Coluna de exclusão de tamanho </td><td> GE Healthcare </td><td> Giro Armadilha G-25 </td></tr><tr><td> Com fundo de vidro câmara de 8-bem </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Microscópio de fluorescência invertido </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> UV fonte de luz LED </td><td> Rapp Opto Eletrônica </td><td> UVILED </td></tr><tr><td> Disco giratório Confocal </td><td> Yokogawa Electric Corp </td><td> CSU10 </td></tr><tr><td> CCD da câmera </td><td> Hamamatsu Photonics </td><td> ORCA-ER </td></tr><tr><td> Da Objectiva 100x </td><td> Zeiss </td><td> Apochromat Plano </td></tr></tbody></table> Esquema sintético do crb (Copyright ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Esquema 1" /></a> Condições sintéticas: (a) BnBr, K 2 CO 3, DMF (b) f.HNO 3, AcOH (c) TFA (d) propargil-Br, K 2 CO 3, DMF, (e) gato glicerol,. PTSA, tolueno (f) NaBH3CN, TiCl4, MeCN (g) Tf 2 O, piridin (h) de metanol HCl, (i) LiAlH 4, THF (j) TsCl, piridina, CH 2 Cl 2 (k) NaN 3, DMF (l) 8, 2,6-di-t-butilpiridina, 2 CH Cl 2 (m) 13, CuSO4, ascorbato, 2-propanol, H2O, CH 2 Cl 2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Regulação dinâmica da família Rho de trifosfatases guanosina pequenas (GTPases) com precisão espaço-temporal grande é essencial para várias funções celulares e eventos 1, 2. A sua natureza dinâmica spatiotemporally foi revelada pela visualização da sua actividade e localização em tempo real 3. A fim de obter uma compreensão mais profunda dos seus papéis em diversas funções celulares a nível molecular, o próximo passo deve ser perturbação das actividades de proteína a uma localização precisa subcelular e de temporização. Para atingir este objectivo, desenvolvemos um método para a induzida pela luz, num espaço-temporal activação controlada de GTPases pequenas pela combinação de duas técnicas: (1) rapamicina induzida heterodimerização FKBP-FRB e (2) um método de foto-enjaulamento de rapamicina. Com o uso de rapamicina mediada heterodimerização FKBP FRB-, desenvolvemos um método para rapidamente induzível activação ou inactivação de pequeno GTPases including Rac 4, Cdc42 4, RhoA 4 e 5 Ras, em que a rapamicina induz a translocação de FKBP-fundidos GTPases, ou os seus activadores, para a membrana plasmática, onde está ancorada FRB. Para o acoplamento com o presente sistema de heterodimerização, também desenvolvido um sistema foto-enjaulamento de análogos de rapamicina. Um composto de foto-engaiolados é uma molécula pequena, cuja actividade é suprimida com um grupo fotocliváveis ​​proteger conhecido como um grupo de encarceramento. Para suprimir a actividade heterodimerização completamente, foi elaborado um rapamicina engaiolados que está preso a uma macromolécula tal que o complexo resultante grande não pode atravessar a membrana plasmática, levando a virtualmente nenhum fundo actividade como um dimerizer química no interior das células 6. A Figura 1 ilustra um esquema do nosso sistema. Com a combinação destes dois sistemas, é localmente recrutado um activador Rac para a membrana plasmática num espaço de tempo de segundos, e conseguido induzida pela luz de activação Rac no lev subcelularel 6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Um método para espácio-temporal controlo da actividade GTPase pequena pela luz é descrito. Este método baseia-se em rapamicina induzida heterodimerização FKBP-FRB e foto-enjaulamento sistemas. Otimização de luz irradiação permite a ativação espaço-temporalmente controlada de pequenas GTPases no nível subcelular.

Espaço-Temporal Manipulação de Atividade GTPase Pequeno em nível subcelular e em escala de tempo de segundos em células vivas

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.7K visualizações.  Johns Hopkins University. Um método para espácio-temporal controlo da actividade GTPase pequena pela luz é descrito. Este método baseia-se em rapamicina induzida heterodimerização FKBP-FRB e foto-enjaulamento sistemas. Otimização de luz irradiação permite a ativação espaço-temporalmente controlada de pequenas GTPases no nível subcelular.

Vídeo: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imagem Lifetime fluorescência de rotores moleculares em células vivas

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioengenharia

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medir as propriedades mecânicas das células vivas Usando Microscopia de Força Atômica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Controle quantitativo e temporal de oxigênio Microenvironment no Ilhéu Individual

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Biossensores raciométrica que medem estado redox mitocondrial e ATP em células vivas de levedura

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento de células do cólon tumor primário Humanos a partir de tecidos cirúrgicos e cultivando-as Diretamente macias elásticas Substratos para Traction Citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

A técnica de livre-Label para a imagem espaço-temporal do Secreções única célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Controle óptico de células vivas atividade elétrica por conjugados Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura de Vida Microbiome com Intestinal Microengineered Humano Villi num Gut-on-a-Chip Microfluidic Dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Medição da dinâmica de força-sensível da proteína em células vivas, usando uma combinação de técnicas fluorescentes

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...