Questo lavoro è stato sostenuto da fondi di dotazione della West Virginia University Mary Babb Randolph Cancer Center. Ringraziamo Susette Mueller (Georgetown University) e Laura Kelley per la consulenza precoce e assistenza. L&#39;uso dello strumento di imaging Virginia Occidentale Microscopia University (supportato dal Babb Mary Randolph Cancer Center e, sovvenzioni NIH RR16440 P20, P30 e P30 RR032138 GM103488) è riconosciuto con gratitudine.

<ol>
	<li>Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+at+the+leading+edge.">Life at the leading edge.</a> Cell. 145, 1012-1022 (2011).</li><li>Murphy, D. A., Courtneidge, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'ins'+and+'outs'+of+podosomes+and+invadopodia:+characteristics,+formation+and+function.">The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function.</a> Nat. Rev. Mol. Cell Biol. 12, 413-426 (2011).</li><li>Linder, S., Wiesner, C., Himmel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degrading+Devices:+Invadosomes+in+Proteolytic+Cell+Invasion.">Degrading Devices: Invadosomes in Proteolytic Cell Invasion.</a> Annu. Rev. Cell. Dev. Biol. , (2010).</li><li>Chen, W. T., Chen, J. M., Parsons, S. J., Parsons, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+degradation+of+fibronectin+at+sites+of+expression+of+the+transforming+gene+product+pp60src.">Local degradation of fibronectin at sites of expression of the transforming gene product pp60src.</a> Nature. 316, 156-158 (1985).</li><li>Artym, V. V., Zhang, Y., Seillier-Moiseiwitsch, F., Yamada, K. M., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interactions+of+cortactin+and+membrane+type+1+matrix+metalloproteinase+at+invadopodia:+defining+the+stages+of+invadopodia+formation+and+function.">Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.</a> Cancer Res. 66, 3034-3043 (2006).</li><li>Ayala, I., 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=Multiple+regulatory+inputs+converge+on+cortactin+to+control+invadopodia+biogenesis+and+extracellular+matrix+degradation.">Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation.</a> J. Cell Sci. , (2008).</li><li>Ammer, A. G., 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=Saracatinib+impairs+head+and+neck+squamous+cell+carcinoma+invasion+by+disrupting+invadopodia+function.">Saracatinib impairs head and neck squamous cell carcinoma invasion by disrupting invadopodia function.</a> J. Cancer Sci. Ther. 1, 52-61 (2009).</li><li>Seals, D. F., 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=The+adaptor+protein+Tks5/Fish+is+required+for+podosome+formation+and+function,+and+for+the+protease-driven+invasion+of+cancer+cells.">The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells.</a> Cancer Cell. 7, 155-165 (2005).</li><li>Yamaguchi, H., 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=Molecular+mechanisms+of+invadopodium+formation:+the+role+of+the+N-WASP-Arp2/3+complex+pathway+and+cofilin.">Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin.</a> J. Cell Biol. 168, 441-452 (2005).</li><li>Kopp, P., 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=The+kinesin+KIF1C+and+microtubule+plus+ends+regulate+podosome+dynamics+in+macrophages.">The kinesin KIF1C and microtubule plus ends regulate podosome dynamics in macrophages.</a> Mol. Biol. Cell. 17, 2811-2823 (2006).</li><li>Oser, M., 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=Cortactin+regulates+cofilin+and+N-WASp+activities+to+control+the+stages+of+invadopodium+assembly+and+maturation.">Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation.</a> J. Cell. Biol. 186, 571-587 (2009).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36-42 (2004).</li><li>Kelley, L. C., 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=Oncogenic+Src+requires+a+wild-type+counterpart+to+regulate+invadopodia+maturation.">Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation.</a> J. Cell Sci. 123, 3923-3932 (2010).</li><li>Chen, W. T., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+is+not+present+in+the+focal+adhesions+formed+between+normal+cultured+fibroblasts+and+their+substrata.">Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata.</a> Proc. Natl. Acad. Sci. U.S.A. 77, 7318-7322 (1980).</li><li>Chen, W. T., Olden, K., Bernard, B. A., Chu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transformation-associated+protease(s)+that+degrade+fibronectin+at+cell+contact+sites.">Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites.</a> J. Cell Biol. 98, 1546-1555 (1984).</li><li>Bharti, S., 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=Src-dependent+phosphorylation+of+ASAP1+regulates+podosomes.">Src-dependent phosphorylation of ASAP1 regulates podosomes.</a> Mol. Cell Biol. 27, 8271-8283 (2007).</li><li>Albrechtsen, R., Stautz, D., Sanjay, A., Kveiborg, M., Wewer, U. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+engagement+of+ADAM12+induces+clusters+of+invadopodia+with+localized+ectodomain+shedding+activity.">Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity.</a> Exp. Cell Res. 317 (10), 195-209 (2011).</li><li>Scott, R. W., 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=kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell Biol. 191, 169-185 (2010).</li><li>Mueller, S. C., Yeh, Y., Chen, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+phosphorylation+of+membrane+proteins+mediates+cellular+invasion+by+transformed+cells.">Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells.</a> J. Cell. Biol. 119, 1309-1325 (1992).</li><li>Bowden, E. T., Coopman, P. J., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invadopodia:+unique+methods+for+measurement+of+extracellular+matrix+degradation+in+vitro.">Invadopodia: unique methods for measurement of extracellular matrix degradation in vitro.</a> Methods Cell Biol. 63, 613-627 (2001).</li><li>Baldassarre, M., 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=Dynamin+participates+in+focal+extracellular+matrix+degradation+by+invasive+cells.">Dynamin participates in focal extracellular matrix degradation by invasive cells.</a> Mol. Biol. Cell. 14, 1074-1084 (2003).</li><li>Alexander, N. R., 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=Extracellular+matrix+rigidity+promotes+invadopodia+activity.">Extracellular matrix rigidity promotes invadopodia activity.</a> Curr. Biol. 18, 1295-1299 (2008).</li><li>Artym, V. V., Yamada, K. M., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+degradation+assays+for+analyzing+local+cell+invasion.">ECM degradation assays for analyzing local cell invasion.</a> Methods Mol. Biol. 522, 211-219 (2009).</li><li>Schoumacher, M., Goldman, R. D., Louvard, D., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin,+microtubules,+and+vimentin+intermediate+filaments+cooperate+for+elongation+of+invadopodia.">Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia.</a> J. Cell Biol. 189, 541-556 (2010).</li><li>Clark, E. S., Whigham, A. S., Yarbrough, W. G., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortactin+is+an+essential+regulator+of+matrix+metalloproteinase+secretion+and+extracellular+matrix+degradation+in+invadopodia.">Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia.</a> Cancer Res. 67, 4227-4235 (2007).</li><li>Yamaguchi, H., 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=Phosphoinositide+3-kinase+signaling+pathway+mediated+by+p110alpha+regulates+invadopodia+formation.">Phosphoinositide 3-kinase signaling pathway mediated by p110alpha regulates invadopodia formation.</a> J. Cell Biol. 193, 1275-1288 (2011).</li><li>Li, 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=The+actin-bundling+protein+fascin+stabilizes+actin+in+invadopodia+and+potentiates+protrusive+invasion.">The actin-bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion.</a> Curr. Biol. 20, 339-345 (2010).</li><li>Yamaguchi, H., 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=Phosphatidylinositol+4,5-bisphosphate+and+PIP5-kinase+Ialpha+are+required+for+invadopodia+formation+in+human+breast+cancer+cells.">Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Ialpha are required for invadopodia formation in human breast cancer cells.</a> Cancer Sci. , (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

La possibilità di visualizzare le cellule che degradano la matrice extracellulare ha aiutato a scoprire i meccanismi molecolari impiegati nelle prime fasi di invasione cellulare. Lanciato da Wen-Tien Chen nei primi anni 1980 4,14,15, rivestimento proteine ​​fluorescenti marcate extracellulari su vetrini per la successiva analisi al microscopio è emerso come la tecnica principale nella valutazione della funzione invadopodia in una vasta gamma di tipi di cellule. Il protocollo prescritto viene illustrato ...

<table border="1"><tbody><tr><td> Nome del reagente / strumento </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> gelatina </td><td> Sigma </td><td> G1890 </td><td> Suini pelle </td></tr><tr><td> saccarosio </td><td> Pescatore </td><td> BP220 </td><td></td></tr><tr><td> Coprioggetto 12 millimetri </td><td> Pescatore </td><td> 50-121-5159 </td><td></td></tr><tr><td> Piastre da 24 pozzetti </td><td> Pescatore </td><td> 08-772-1 </td><td> BD Falcon </td></tr><tr><td> acido nitrico </td><td> Ricca </td><td> R5326000 </td><td> Soluzione al 20% </td></tr><tr><td> poli-L-lisina </td><td> Scienze della microscopia elettronica </td><td> 19320-B </td><td> 0,1% soluzione </td></tr><tr><td> glutaraldeide </td><td> Sigma </td><td> G7526 </td><td> Soluzione 8% </td></tr><tr><td&gt; Oregon verde 488-coniugato gelatina </td><td> Invitrogen </td><td> G-13186 </td><td></td></tr><tr><td> sodio boroidruro </td><td> Sigma </td><td> 213462 </td><td></td></tr><tr><td> Triton X-100 </td><td> Pescatore </td><td> BP151 </td><td></td></tr><tr><td> rodamina-falloidina </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> anti-cortactin (clone 4F11) </td><td> Millipore </td><td> 05-180 </td><td></td></tr><tr><td> Anticorpo anti-FLAG </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> Alexa Fluor 647 di capra anti-IgG di topo </td><td> Invitrogen </td><td> A21235 </td><td></td></tr><tr><td> Prolong antifade Oro </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> LSM 510 microscopio confocale </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ software </td><td> Di dominio pubblico </td><td></td><td> <a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a> </td></tr><tr><td> AutoQuant X2.2 software </td><td> Media Cibernetica </td><td></td><td></td></tr><tr><td> NIS Elements </td><td> Nikon </td><td></td><td></td></tr></tbody></table>

quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts

Invasione cellulare nei tessuti locali è un processo importante nello sviluppo e omeostasi. Invasione Malregulated e il movimento delle cellule successiva è caratteristica di molteplici processi patologici, tra cui l&#39;infiammazione, le malattie cardiovascolari e le metastasi delle cellule tumorali 1. Degradazione proteolitica focalizzata di matrice extracellulare (ECM), componenti della membrana basale epiteliale o endoteliale è un passo fondamentale nel dare inizio invasione cellulare. Nelle cellule tumorali, ampia analisi in vitro ha determinato che il degrado ECM si ottiene ventrali actina ricchi di strutture a membrana protrusione chiamati invadopodia 2,3. Forma Invadopodia in stretta apposizione della ECM, dove moderare ripartizione ECM attraverso l&#39;azione di metalloproteinasi della matrice (MMP). La capacità delle cellule tumorali di formare invadopodia correla direttamente con la capacità di invadere stroma locale e componenti associati vascolari 3. _content &quot;&gt; Visualizzazione di invadopodia-mediata degradazione ECM delle cellule mediante microscopia a fluorescenza con colorante marcati con proteine ​​della matrice rivestite su vetro coprioggetti è emerso come la tecnica più diffusa per valutare il grado di proteolisi matrice e cellulari potenziale invasivo 4,5. Qui descriviamo una versione del metodo standard per generare vetrini fluorescenza etichettati utilizzando un comune Oregon verde-488 coniugato gelatina. Questo metodo è facilmente scalata per produrre rapidamente un elevato numero di vetrini coprioggetto rivestiti. Mostriamo alcuni degli artefatti comuni microscopici che spesso si incontrano durante questa procedura e come questi possono essere evitati. Infine, si descrivono metodi standardizzati software prontamente disponibile per consentire la quantificazione della degradazione della matrice gelatinosa etichettati mediata da cellule singole e intere popolazioni cellulari. Le procedure descritte consentono di monitorare accurato e riproducibile invadopodiaattività, e può anche servire come una piattaforma per la valutazione dell&#39;efficacia di espressione della proteina modulante o prove di anti-invasivi composti sulla degradazione della matrice extracellulare in impostazioni singole e multicellulari.

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Descriviamo il metodo di prototipo per la produzione di vetrini al microscopio a fluorescenza rivestite con gelatina per la visualizzazione invadopodia-mediata degradazione della matrice. Tecniche di calcolo utilizzando il software disponibile sono presentati per quantificare i livelli risultanti di proteolisi matrice di singole cellule all&#39;interno di una popolazione mista e per gruppi multicellulari che comprende interi campi microscopici.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

quantitative-measurement-invadopodia-mediated-extracellular-matrix

Research

JoVE Journal

Biology

19.1K Views.  West Virginia University. Descriviamo il metodo di prototipo per la produzione di vetrini al microscopio a fluorescenza rivestite con gelatina per la visualizzazione invadopodia-mediata degradazione della matrice. Tecniche di calcolo utilizzando il software disponibile sono presentati per quantificare i livelli risultanti di proteolisi matrice di singole cellule all'interno di una popolazione mista e per gruppi multicellulari che comprende interi campi microscopici.

Video: Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Studiare proteolisi della ciclina B a livello di singola cellula in popolazioni di cellule intere

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Ricerca

Biologia

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Base-Probe Real-time PCR Approcci per la misurazione quantitativa dei microRNA

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Misurazione della extracellulare Ion Flussi Utilizzando il ionoselettivo autoreferenziale microelettrodo Tecnica

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Arricchimento della matrice extracellulare proteine ​​da tessuti e digestione in Peptidi per l&#39;analisi Spettrometria di Massa

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Efficiente Espressione cellulari di mammifero e Passo singolo Purificazione di extracellulare glicoproteine ​​per cristallizzazione

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Scanning Electron Microscopy di tessuto macerato di visualizzare la matrice extracellulare

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

A Rapid, metodo scalabile per l&#39;isolamento, studio funzionale, e analisi di Cell-derivati ​​matrice extracellulare

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...