Name: single-cell microinjection for cell communication analysis
Uploaded: 2017-02-26T13:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

<html> 1. Preparação de células <ol><li> Manter uma cultura de uma linha celular epitelial tímica (IT76M1) ou célula a ser testada numa incubadora (37 ° C / 5% de CO 2). </li><li> Lave as células com PBS 1x (repetir este item 3x). </li><li> Adicionar tripsina às células durante 5 min. </li><li> Adicionar meio (o dobro do volume de tripsina adicionada no ponto 1.3) com 10% de FBS (soro fetal bovino) para as células com tripsina e centrifugação (800 xg durante 5 min). </li><li> Contar as células num hemocitómetro. </li><li> Ajustar a densidade de células de acordo com o tipo de células como as células têm que estar em estreito contacto umas com as outras para permitir o acoplamento. Nota: No nosso caso, usamos 3 x 10 5 células por 35 milímetros placa de Petri. </li></ol> 2. Preparação Micropipeta <ol><li> Puxar a micropipeta, conforme especificado a partir de uma micropipeta capilar de vidro (1,5 mm de diâmetro) para uma final de 0,2 um de diâmetro de modo a atingir uma resistência final de aproximadamente 30 mohmsf &quot;&gt; 17, 18. NOTA: Alternativamente, as pipetas de injecção pode ser comprado. A resistência depende do tamanho da célula, por exemplo, seria necessário para células acinares pancreáticas um microeléctrodo resistência mais elevada, por exemplo (100-150 mohms) 19. Um problema comum é que pode ocorrer a precipitação da solução amarelo Lucifer, que pode, em seguida, obstruir a micropipeta e pode necessitar de filtração ou centrifugação antes. Antes da injecção, a micropipeta devem ser analisadas sob o microscópio para detectar se existe uma obstrução ou qualquer tipo de interrupção 13. A micropipeta pode ser testada através da injecção de LY com a ponta da micropipeta dentro de uma solução salina. </li></ol> 3. Teste a micropipeta <ol><li> Preparar a solução de Amarelo Lúcifer (5%) em 150 mmol / L de LiCl e carregar a micropipeta com uma seringa ou por enchimento (colocar nele LY solução). </li><li> Coloque a micropipette ao longo de 35 mm placa de Petri com as células IT76M1 na estação de trabalho microinjecção e submergir a ponta da micropipeta de vidro para o meio celular. Concentre-se na micropipeta e executar um teste de corante fluindo através da aplicação de um pulso. </li></ol> 4. uma única célula Lucifer Yellow Microinjeção <ol><li> Concentre-se o microscópio logo acima da camada de células usando um grande aumento fi cação (40X) e desça lentamente a pipeta para as células usando o micromanipulador. </li><li> Perfurar a célula alvo quando a ponta está perto o suficiente para tocar a membrana celular, e aplique uma pequena pulsação de hiperpolarização para introduzir o LY na célula. A tensão aplicada dependerá da carga líquida do corante a ser injectada. Alternativamente, alguns outros corantes podem ser usados ​​com esta técnica, como mostrado na Tabela 1. Nota: Em princípio, qualquer corante hidrofílico com um PM inferior a 1 kDa poderia ser usada. No entanto, a taxa de transferência pode variar de acordo com o peso e hidrofilicidade. Além disso, utransferência nspecific do corante utilizado deve ser avaliada. </li><li> Capturar imagens de células 3 min após a injeção de corante ou fazer um pequeno filme com microscopia de lapso de tempo (30 fps). NOTA: Uma abordagem semelhante pode ser visto na Hitomi et al (2015) 20. Para evitar a comunicação por pontes intercelulares (mitose incompleto), um co-injecção de dextrano rodamina (de 2 a 10 kDa), que não passa através de junções de hiato, mas passa através de pontes intercelulares e certos tipos de nanotubules é recomendada como mostrado na Figura 2 . </li></ol>

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The authors have no conflicts of interest.

A fim de verificar a presença de junções de hiato funcionais intercelular, a utilização de marcadores, que são membrana impermeável, embora permeável por meio de canais intercelulares são necessários 16. Fluoresceína, o primeiro corante fluorescente para observar célula-a-célula de acoplamento 22, é permeável entre membranas não juncionais 3 e, por conseguinte, tem sido substituído por Lúcifer corante Amarelo 15.<...

As junções de hiato são canais intercelulares que permitem a intercomunicação entre células vizinhas 1. Esta comunicação conecta duas ou mais células vizinhas, onde cada um contribui com um connexon ou hemichannel para formar o canal intercelular. Em células de mamíferos, o connexon é formado por seis conexinas, monómeros com quatro domínios transmembranares e um terminal de C e N dentro do citoplasma 2. As junções de hiato não só permitir o fluxo de iões, segundos mensageiros e pequenos metabolitos, mas também contribuir para muitas formas de comunicação celular em muitos processos fisiológicos, tais como a transmissão sináptica, a contracção cardíaca, crescimento e diferenciação celular 3, 4, 5, 6, 7, 8. Além disso as junções de hiato foram associados commuitas doenças incluindo cancro 9, 10, 11, atrofia muscular, algumas doenças genéticas e doenças desmielinizantes 12. Este tipo de interferência intercelular pode ser avaliada através de vários métodos 13, 14, 15, 16. Neste artigo, vamos mostrar como realizar uma microinjeção de uma única célula de Lucifer Yellow visualizar comunicação celular via gap-junções em células vivas. Discute-se como preparar as células e a micropipeta, a utilização do micromanipulador e a injecção do corante Amarelo Lúcifer em uma linha de células epiteliais do timo. Normalmente, este procedimento experimental poderia ser analisados ​​pela média de células ligadas à célula carregado com corante. Além disso, este método pode ser usado com outros corantes fluorescentes com peso molecular inferior a lacunajunções de corte, que é cerca de 1.000 daltons.

<table><tbody><tr><td>Lucifer yellow</td><td>Sigma</td><td>L0259</td><td></td></tr><tr><td>Lithium Chloride</td><td>Sigma</td><td>L4408</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma&amp;nbsp;</td><td>P4417</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R4130</td><td></td></tr><tr><td>Bovine fetal serum</td><td>Cultilab</td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4799</td><td></td></tr><tr><td>vibration-insulated table&amp;nbsp;</td><td>Newport</td><td>VH3036W-OPT</td><td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MMO-203</td><td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td></tr><tr><td>Current Generator&amp;nbsp;</td><td>Digitimer</td><td>DS2</td><td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

single-cell microinjection for cell communication analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Tímico linha celular epitelial de TI-76MI foram usadas para avaliar corante de acoplamento por junções de hiato como estas células foram descritos para expressar junções de hiato funcionais formados por conexina 43 21. A Figura 1 mostra a injecção de Amarelo Lúcifer quando aplicado na uma célula abaixo da ponta da pipeta. Após alguns minutos, as células tornam-se ligados fluorescente (asteriscos), indicando a difusão do corante fluorescente através d...

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis

Descrevemos aqui como executar uma microinjeção de uma única célula de Lucifer Yellow visualizar comunicação celular via gap-junções em células vivas, e fornecer algumas dicas úteis. Esperamos que este trabalho irá ajudar a todos para avaliar o grau de acoplamento celular devido à junções comunicantes funcionais. Tudo o descrito aqui pode ser, em princípio, adaptado para outros corantes fluorescentes com peso molecular inferior a 1.000 Daltons.

Microinjeção de uma única célula para análise da comunicação celular

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

single-cell-microinjection-for-cell-communication-analysis

Research

JoVE Journal

Biology

11.2K visualizações.  Oswaldo Cruz Foundation.  Descrevemos aqui como executar uma microinjeção de uma única célula de Lucifer Yellow visualizar comunicação celular via gap-junções em células vivas, e fornecer algumas dicas úteis. Esperamos que este trabalho irá ajudar a todos para avaliar o grau de acoplamento celular devido à junções comunicantes funcionais. Tudo o descrito aqui pode ser, em princípio, adaptado para outros corantes fluorescentes com peso molecular inferior a 1.000 Daltons. 

Vídeo: Single-cell Microinjection for Cell Communication Analysis

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Análise do transcriptoma de uma única célula

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neurociência

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Um chip microfluídico para a Versátil Análise Química de células individuais

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Bioengenharia

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Intranucleares microinjeção de DNA em Dissociated neurônios de mamíferos adultos

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Biologia

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análise de Cinética mRNA Nuclear Exportação em células de mamíferos por microinjeção

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Microscópio óptico e Eletrônica (CLEM) como uma ferramenta para visualizar moléculas microinjetados e seus eucarióticas Sub-celulares Metas


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Manipulação de células-tronco neurais únicas e neurônios em fatias cerebrais usando microinjeção robótica

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Análise da localização de snRNA Spliceosomal em células HeLa humanas usando microinjeção

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microinjeções de ouriço do mar zigotos

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Técnicas de micromanipulação, permitindo a análise da dinâmica morfogenética e rotatividade dos reguladores do citoesqueleto

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

Source: Catrina, I. E., et al. <a href="https://www.jove.com/video/58545/" target="_blank">Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers</a>. J. Vis. Exp. (2019).
Due to their large size and relatively simple organization, Drosophila nurse cells are ideally suited for live cell imaging studies. This video describes a microinjection method for exogenous compounds delivery into the cells, and the featured protocol demonstrates the procedure with fluorescent oligonucleotide probes called molecular beacons (MBs) used to visualize endogenous mRNA transcripts.

Microinjeção de células enfermeiras de Drosophila: um método de entrega intracelular

Source: Catrina, I. E., et al. Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers. J. Vis. Exp. (2019).
Due to their ...