Os autores gostariam de reconhecer o Dr. Kees Jalink (Instituto de Câncer dos Países Baixos e o Centro de Microscopia Avançada van Leeuwenhoek, Amsterdã, Holanda) por nos fornecer o biosensor H188 cAMP FRET e Kenny Trinh (College of Engineering, University of South Alabama) para ajuda técnica na redução do tempo de tempo que leva para executar nossos scripts de programação desenvolvidos sob medida.
Os autores gostariam de reconhecer as fontes de financiamento: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+adenosine+3':5'-monophosphate+and+adenosine+3':5'-monophosphate-dependent+protein+kinase+in+heart+tissue.">Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.</a> The Journal of Biological Chemistry. 252, 3854-3861 (1977).</li><li>Terrin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGE1+stimulation+of+HEK293+cells+generates+multiple+contiguous+domains+with+different+[cAMP]:+role+of+compartmentalized+phosphodiesterases.">PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases.</a> The Journal of Cell Biology. 175, 441-451 (2006).</li><li>Bacskai, B. 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Os Drs. Leavesley e Rich revelam interesse financeiro em uma empresa iniciante, SpectraCyte, LLC, que foi formada para comercializar tecnologias de imagem espectral. No entanto, todos os procedimentos descritos neste protocolo foram realizados utilizando produtos comercialmente disponíveis não associados à SpectraCyte, LLC.

O desenvolvimento de biosensores FRET permitiu a medição e visualização de sinais nucleotídeos cíclicos em células únicas, e há grande promessa de visualização de eventos de sinalização subcelular13,22,37,38. No entanto, o uso de biosensores FRET apresenta várias limitações, incluindo as características de baixo sinal-ruído de muitos repórteres fret à base de proteína fluor...

Monofosfato de adenosina cíclica (cAMP) é um segundo mensageiro envolvido em processos celulares e fisiológicos chave, incluindo divisão celular, influxo de cálcio, transcrição genética e transdução de sinais. Um conjunto crescente de evidências sugere a existência de compartimentos cAMP na célula através dos quais a especificidade de sinalização é alcançada1,2,3,4,5,6,7. Até recentemente, a compartimentação cAMP era inferida com base em efeitos fisiológicos ou celulares distintos induzidos por diferentes agonistas receptores acopladosG 8,9,10,11. Mais recentemente, as sondas de imagem de fluorescência baseadas em FRET forneceram novas abordagens para a medição direta e observação de sinais cAMP em uma célula12,13,14.
A transferência de energia de ressonância förster (FRET) é um fenômeno físico no qual ocorre a transferência de energia entre moléculas doadoras e aceitadoras de forma não radiativa quando as moléculas estão próximas15,16. Com o desenvolvimento de indicadores fluorescentes baseados em FRET, esse fenômeno físico tem sido utilizado em aplicações biológicas para estudar interações proteína-proteína17, co-localização proteica18, Ca+2 sinalizando19, expressão genética20,divisão celular21 e sinalização nucleotídea cíclica. Os indicadores cAMP baseados em FRET normalmente consistem em um domínio de vinculação cAMP, um fluoróforo doador e um fluoróforoaceitor 22. Por exemplo, o sensor H188 cAMP12,22 utilizado nesta metodologia consiste em um domínio de ligação cAMP obtido da Epac, sanduíche entre fluoroforos turquesa (doador) e Vênus (aceitador). Em condições basais (sem entrada), Turquesa e Vênus estão em uma orientação de tal forma que o FRET ocorre entre os fluoroforos. Ao vincular o cAMP ao domínio de ligação, ocorre uma alteração conformacional de tal forma que Turquesa e Vênus se afastam, resultando em uma diminuição do FRET.
As abordagens de imagem baseadas em FRET oferecem uma ferramenta promissora para investigar e visualizar sinais cAMP dentro de uma célula. No entanto, as técnicas atuais de imagem microscópica baseadas em FRET são muitas vezes apenas parcialmente bem sucedidas em alcançar força de sinal suficiente para medir o FRET com clareza espacial subcelular. Isso se deve a vários fatores, incluindo a intensidade limitada do sinal de muitos repórteres da FRET, o alto nível de precisão necessário para quantificar com precisão as mudanças na eficiência do FRET e a presença de fatores de confusão, como a autofluorescência celular23,24. O resultado é muitas vezes uma imagem FRET que é atormentada por SNR fraco, tornando muito difícil a visualização de mudanças subcelulares no FRET. Além disso, a investigação de sinais cAMP espacialmente localizados tem sido realizada quase exclusivamente em apenas duas dimensões espaciais e a distribuição axial cAMP raramente foi considerada25. Isso é provável porque o SNR baixo impediu a capacidade de medir e visualizar gradientes cAMP em três dimensões espaciais. Para superar as limitações do uso de sensores FRET com baixo SNR, implementamos abordagens hiperespectrais de imagem e análise para medir o FRET em células únicas25,26,27.
Abordagens de imagem hiperespectral foram desenvolvidas pela NASA para diferenciar objetos terrestres presentes em imagens de satélite28,29. Essas técnicas foram traduzidas para o campo de microscopia de fluorescência30,com vários sistemas comerciais de microscópio confocal oferecendo detectores espectrais. Em imagens tradicionais (não espectrais) de fluorescência, a amostra é animada usando um filtro de passe de banda ou uma linha laser, e a emissão é coletada usando um segundo filtro de passe de banda, muitas vezes selecionado para corresponder ao comprimento de onda de emissão máxima do fluoróforo(s). Em contrapartida, as abordagens de imagem hiperespectral buscam amostrar um perfil espectral completo da emissão de fluorescência26,31,32 ou excitação33,34 em intervalos específicos de comprimento de onda. Em nossos estudos anteriores, mostramos que as abordagens de imagem e análise hiperespectral podem oferecer uma quantificação melhorada dos sinais DE TRAC nas células quando comparadas às técnicas tradicionais de imagem FRET baseadas emfiltros 26. Aqui, apresentamos uma metodologia para a realização de imagens e análises hiperespectrais de FRET hiperdimensionais (x, y, z e λ) para medir e visualizar distribuições cAMP em três dimensões espaciais. Essas abordagens permitiram a visualização de gradientes espaciais cAMP induzidos por agonista em célulasúnicas 25. Curiosamente, dependendo do agonista, gradientes cAMP podem ser aparentes nas células. A metodologia aqui apresentada utiliza o descompreamento espectral de fundo não uniforme e autofluorescência celular para melhorar a precisão das medições de FRET. Embora essa metodologia seja demonstrada em células endoteliais microvasculares pulmonares (PMVECs) utilizando um biosensor CAMP FRET, a metodologia poderia ser facilmente modificada para uso com repórteres fret alternativos ou linhas celulares alternativas.

<table>
	<tbody>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
			<td>Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Solvent used to prepare stock solution forskolin.</td>
		</tr>
		<tr>
			<td>DRAQ5 Fluoroscent Probe Solution</td>
			<td>Thermo Scientific</td>
			<td>62252</td>
			<td>Nuclear label</td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Contains nutrients and growth factors for the cells to grow and divide in the culture dishes.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F6178</td>
			<td>Growth factor suppliment that is added to culture medium, DMEM</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F3917</td>
			<td>Adenyly cyclase activator.</td>
		</tr>
		<tr>
			<td>H188 Cyclic AMP FRET biosensor</td>
			<td>Netherland Cancer Institute, Dr. K. Jalink</td>
			<td>Gift</td>
			<td>Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>image.net</td>
			<td>Free download</td>
			<td>Another image processing platform used to extact spectral information and image processing.</td>
		</tr>
		<tr>
			<td>Integrating Sphere</td>
			<td>Ocean Optics</td>
			<td>FOIS-1</td>
			<td>Used to measure illumination intensity of the laser line at different laser intensities (?).</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td>Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell.</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Kit</td>
			<td>Invitrogen</td>
			<td>L3000-015</td>
			<td>Transfection reagent used to transfect cells with H188 FRET biosensor</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2019a</td>
			<td>Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>Nikon A1R</td>
			<td>Spectral image acquisition is performed using confocal microscope.</td>
		</tr>
		<tr>
			<td>Nikon Elements Software</td>
			<td>Nikon Instruments</td>
			<td>Software dongle</td>
			<td>used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R</td>
		</tr>
		<tr>
			<td>NIST-Traceable Calibration Lamp</td>
			<td>Ocean Optics</td>
			<td>LS-1-CAL-INT</td>
			<td>A lamp with a known spectrum for use as a standard</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>coomonly used buffer suring cell culture</td>
		</tr>
		<tr>
			<td>Pulmonary Microvascular Endothelial Cells (PMVECs)</td>
			<td>In house - Cell culture core, Univeristy of South Alabama</td>
			<td>Isolated from Rat pulmonary microvasculature</td>
			<td>PMVECs form inner lining of a blood vessel.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/ml)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>antibiotics are added to culture medum to prevent contamination of the cells.</td>
		</tr>
		<tr>
			<td>Pre-Cleaned Gold Seal Micro Slides</td>
			<td>Clay Adams</td>
			<td>3010</td>
			<td>Microscope slides used for cell fixation</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mounting Media</td>
			<td>Invitrogen</td>
			<td>P36961</td>
			<td>If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading.</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65000</td>
			<td>Used to measure spectral response of the light source (?)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating.</td>
		</tr>
		<tr>
			<td>Tyrodes Buffer</td>
			<td>Made in-house</td>
			<td>Made in-house</td>
			<td>Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition.</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well.</td>
		</tr>
		<tr>
			<td>25 mm Round Microscope Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12545102</td>
			<td>Cells were grown on round glass coverslips</td>
		</tr>
		<tr>
			<td>60X Ojective</td>
			<td>Nikon Instruments</td>
			<td>Plan Apo VC 60X/1.2 WI &#8734;/0.15-0.18 WD 0.27</td>
			<td>water immersion and commonly used objective for cells</td>
		</tr>
	</tbody>
</table>

measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis

O AMP cíclico é um segundo mensageiro que está envolvido em uma ampla gama de atividades celulares e fisiológicas. Vários estudos sugerem que os sinais de cAMP são compartimentados, e que a compartimentação contribui para a sinalização da especificidade dentro da via de sinalização cAMP. O desenvolvimento de biosensores baseados em transferência de energia de ressonância Fӧrster (FRET) promoveram a capacidade de medir e visualizar sinais cAMP nas células. No entanto, essas medidas são muitas vezes limitadas a duas dimensões espaciais, o que pode resultar em má interpretação dos dados. Até o momento, houve apenas medições muito limitadas de sinais cAMP em três dimensões espaciais (x, y e z), devido às limitações técnicas no uso de sensores FRET que inerentemente exibem baixa relação sinal/ruído (SNR). Além disso, as abordagens tradicionais de imagem baseadas em filtros são muitas vezes ineficazes para a medição precisa de sinais cAMP em regiões subcelulares localizadas devido a uma série de fatores, incluindo crosstalk espectral, força limitada do sinal e autofluorescência. Para superar essas limitações e permitir que os biosensores baseados em FRET sejam usados com fluoroforos múltiplos, desenvolvemos abordagens hiperespectrais de imagem e análise de FRET que fornecem especificidade espectral para calcular a eficiência do FRET e a capacidade de separar espectralmente os sinais FRET de confundir autofluorescência e/ou sinais de rótulos fluorescentes adicionais. Aqui, apresentamos a metodologia de implementação de imagens FRET hiperespectrais, bem como a necessidade de construir uma biblioteca espectral apropriada que não seja subamostra nem supersamplada para realizar descomprturações espectrais. Enquanto apresentamos essa metodologia para medição de distribuições tridimensionais de cAMP em células endoteliais microvasculares pulmonares (PMVECs), essa metodologia poderia ser usada para estudar distribuições espaciais de cAMP em uma gama de tipos celulares.

Este protocolo descreve o uso de abordagens hiperespectrais de imagem e análise de FRET para medir gradientes cAMP em três dimensões espaciais em células vivas. Existem várias etapas fundamentais envolvidas na geração desses resultados, para as quais é necessária uma atenção cuidadosa ao analisar e quantificar os dados. Essas etapas-chave incluem a construção de uma biblioteca espectral apropriada, descompramento espectral de fundo, limiar para identificar bordas celulares e cálculos de eficiência do FRET....

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and ÃŽÂ» Hyperspectral FRET Imaging and Analysis at JoVE.com

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &lambda; Hyperspectral FRET Imaging and Analysis

Devido à relação sinal-ruído inerente (SNR) de sensores baseados em transferência de energia de ressonância (FRET) Fӧrster, a medição dos sinais cAMP tem sido desafiadora, especialmente em três dimensões espaciais. Aqui, descrevemos uma metodologia hiperespectral de imagem e análise de FRET que permite a medição da distribuição cAMP em três dimensões espaciais.

Medição de distribuições 3-dimensionales cAMP em células vivas usando imagens e análises hiperespectrais de FRET 4-Dimensional (x, y, z e λ) Hiperespectrais

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...

measurement-3-dimensional-camp-distributions-living-cells-using-4

Research

JoVE Journal

Biology

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

3.8K visualizações.  University of South Alabama. Devido à relação sinal-ruído inerente (SNR) de sensores baseados em transferência de energia de ressonância (FRET) Fӧrster, a medição dos sinais cAMP tem sido desafiadora, especialmente em três dimensões espaciais. Aqui, descrevemos uma metodologia hiperespectral de imagem e análise de FRET que permite a medição da distribuição cAMP em três dimensões espaciais.

Vídeo: Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and λ Hyperspectral FRET Imaging and Analysis

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulso-chase Análise de Cadeias N-linked Açúcar de Glicoproteínas em células de mamíferos

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Biologia

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

Análise imunocitoquímica de células-tronco humanas pluripotentes usando um Self-Made Aparelho Cytospin

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Medição simultânea em pH endocítica e citossólicos Compartimentos em células vivas usando microscopia confocal

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imagens de fluorescência com um nanômetros Precisão (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Medição das flutuações cálcio dentro do Sarcoplasmic retículo de células musculares lisas cultivadas utilizando FRET baseado em imagem confocal

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...