Questo lavoro è stato sostenuto da una sovvenzione da parte del National Institutes of Health, Istituto Nazionale del Diabete e Malattie Digestive e Renali, 2R01DK59558 (a GN). MSNA Translational Research Institute sostenuto dal National Institutes of Health Centro Nazionale per la Ricerca Risorse concessione UL1 RR029884 fornito un finanziamento parziale per Core Citometria a Flusso di MSNA. Ringraziamo il Dott. Peipei Ping (Università di California a Los Angeles, Los Angeles, CA) per fornire un &#39;aliquota di adenovirus portare cDNA codificante il dominante negativo (inattivo) mutante della PKC-ε e il dottor Allen Samarel (Loyola University Medical Center; Maywood, IL) per fornire una aliquota di vettore adenovirale codificante mutante costitutivamente attivo di PKC-ε. Ringraziamo anche Drs. Peter Parker e Peter Sugden (Imperial College London, Londra, Regno Unito) per la fornitura di cDNA codificante costitutivamente attivi PKC-ε.

<ol>
	<li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+culture+conditions+stimulate+gluconeogenesis+in+primary+cultures+of+renal+proximal+tubule+cells.">Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells.</a> Am. J. Physiol. 268, C1053-C1061 (1995).</li><li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+regulates+growth+and+metabolism+of+renal+cells:+improvements+in+cell+culture.">L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.</a> Am. J. Physiol. 271, 2072-2080 (1996).</li><li>Ping, P., Takano, H., Zhang, J., Tang, X. L., Qiu, Y., Li, R. C., Banerjee, S., Dawn, B., Balafonova, Z., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoform-selective+activation+of+protein+kinase+C+by+nitric+oxide+in+the+heart+of+conscious+rabbits:+a+signaling+mechanism+for+both+nitric+oxide-induced+and+ischemia-induced+preconditioning.">Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.</a> Circ. Res. 84, 587-604 (1999).</li><li>Wotton, D., Ways, D. K., Parker, P. J., Owen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+both+Raf+and+Ras+is+necessary+for+activation+of+transcription+of+the+human+T+cell+receptor+beta+gene+by+protein+kinase+C,+Ras+plays+multiple+roles.">Activity of both Raf and Ras is necessary for activation of transcription of the human T cell receptor beta gene by protein kinase C, Ras plays multiple roles.</a> J. Biol. Chem. 268, 17975-17982 (1993).</li><li>Nowak, G., Bakajsova, D., Samarel, 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=Protein+kinase+C-&#949;+activation+induces+mitochondrial+dysfunction+and+fragmentation+in+renal+proximal+tubules.">Protein kinase C-&#949; activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.</a> Am. J. Physiol. Renal Physiol. 301, F197-F208 (2011).</li><li>Nowak, G., Clifton, G. L., Godwin, M. L., Bakajsova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ERK1/2+pathway+mediates+oxidant-induced+decreases+in+mitochondrial+function+in+renal+cells.">Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.</a> Am. J. Physiol. Renal Physiol. 291, 840-855 (2006).</li><li>Shaik, Z. P., Fifer, E. K., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+activation+improves+oxidative+phosphorylation+in+renal+proximal+tubular+cells+following+nephrotoxicant+injury.">Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury.</a> Am. J. Physiol. Renal Physiol. 294, F423-F432 (2008).</li><li>Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#945;+and+ERK1/2+mediate+mitochondrial+dysfunction,+decreases+in+active+Na++transport,+and+cisplatin-induced+apoptosis+in+renal+cells.">Protein kinase C-&#945; and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells.</a> J. Biol. Chem. 277, 43377-43388 (2002).</li><li>Liu, X., Godwin, M. L., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-+a+inhibits+the+repair+of+oxidative+phosphorylation+after+S-(1,2-dichlorovinyl)-L-cysteine+injury+in+renal+cells.">Protein kinase C- a inhibits the repair of oxidative phosphorylation after S-(1,2-dichlorovinyl)-L-cysteine injury in renal cells.</a> Am. J. Physiol. Renal Physiol. 287, F64-F73 (2004).</li><li>Nowak, G., Price, P. M., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+a+functional+p21WAF1/CIP1+gene+accelerates+caspase-independent+apoptosis+induced+by+cisplatin+in+renal+cells.">Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.</a> Am. J. Physiol. Renal Physiol. 285, F440-F450 (2003).</li><li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+renal+cell+apoptosis:+caspase+3-dependent+and+-independent+pathways.">Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.</a> J. Pharmacol. Exp. Ther. 302, 8-17 (2002).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;approccio qui presentato consente di sovraespressione di isoenzimi singoli PKC nella coltura primaria di cellule del tubulo prossimale renale. Ci sono diversi punti di forza di questo approccio: 1. Permette di studiare meccanismi di regolazione in una popolazione omogenea di cellule (cellule del tubulo prossimale renale) che sono l&#39;obiettivo primario per insulti vari (ischemia, ipossia, stress ossidativo), farmaci e nephrotoxicants all&#39;interno del rene. 2. Funzioni mitocondriali in questo modello in vi...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Cappa a flusso laminare </td><td> Thermo Electron Corporation </td><td> FORMA 1104 </td></tr><tr><td> 2 ml e 15 ml smerigliatrice Dounce tessuto </td><td> WHEATON </td><td> 989-24607, 357.544 </td></tr><tr><td> 85 e 235 micron maglia di nylon </td><td> Piccole parti </td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50 ml provetta sterile </td><td> BIOLOGIX </td><td> BCT-P 50BS </td></tr><tr><td> 1,5 ml micro tubo </td><td> Sarstedt </td><td> 72.690.001 </td></tr><tr><td> 35 x 10 mm piastre di coltura sterili </td><td> Corning </td><td> 430165 </td></tr><tr><td> Jouan Centrifuga </td><td> Jouan </td><td> Jouan CR3 11175704 Rotori: Jouan T40 </td></tr><tr&gt; <td> Regolabile micro-centrifuga </td><td> SIGMA </td><td> Modello 1-15 </td></tr><tr><td> Monitor di ossigeno biologica </td><td> YSI Incorporated </td><td> YSI modello 5300A </td></tr><tr><td> Singolo Ossigeno Sistema Camerale Micro </td><td> YSI Incorporated </td><td> 5356S </td></tr><tr><td> Sonda di ossigeno </td><td> YSI Incorporated </td><td> 5331A </td></tr><tr><td> Bagno di circolazione </td><td> YSI Incorporated </td><td> 5310 </td></tr><tr><td> KCl e Kit membrana standard </td><td> YSI Incorporated </td><td> 5775 </td></tr><tr><td> Agitatore magnetico </td><td> YSI Incorporated </td><td> 5222 </td></tr><tr><td> Registratore Flatbed </td><td> Kipp &amp; Zonen </td><td> BD 11E </td></tr><tr><td> 48 pozzetti e piastre da 96 pozzetti trasparenti </td><td> Costar </td><td> 3548, 3679 </td></tr><tr><td> Thermomixer R </td><td> Eppendorf </td><td> 5355 21919 </td></tr><tr><td> Agitatore orbitale MaxQ 2000 </td><td> Thermo Scientific </td><td> SHKA 2000 </td></tr><tr><td> Spectra FLUOR Plus (assorbanza / fluorescenza / luminescenza lettore) </td><td> Tecan </td><td> F129005 </td></tr><tr><td> Camicia d&#39;acqua degli Stati Uniti Autoflow automatica di CO 2 incubatore </td><td> Nuaire </td><td> NU 4850 </td></tr><tr><td> 12x75 mm in polistirolo Provette di coltura per citometria a flusso </td><td> Fisher Marca </td><td> 14-961-20 </td></tr><tr><td> Axioskop Obiettivo a immersione in acqua 63x / 0,90 W </td><td> Carl Zeiss </td><td> 114846 Achroplan 44 00 67 </td></tr><tr><td> DMEM / F12 </td><td> Cellgro </td><td> 99-830 - PB </td></tr><tr><td> DMEM / Ham F12 </td><td> Sigma </td><td> D 2906 - 1L </td></tr><tr><td> Deferoxamina Mesylate </td><td> Hospira </td><td> D110 </td></tr><tr><td> Collagenasi Tipo I </td><td> Worthington </td><td> 4196 </td></tr><tr><td> Inibitore della tripsina </td><td> Sigma </td><td> T 6522 - 500mg </td></tr><tr><td> 5,5 &#39;, 6,6&#39;-tetracloro-1, 1 &#39;, 3,3&#39;-tetraethylbenzimidazolylcarbocyanine ioduro (JC-1) </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> Mitotracker Red </td><td> Invitrogen </td><td> M22425 </td></tr><tr><td> ATP bioluminescenza Assay Kit HS II </td><td> Roche </td><td> 11 699 709 001 </td></tr><tr><td> Annessina V - FITC soluzione </td><td> BioVision </td><td> 1001 - 200 </td></tr><tr><td> Citometro di flusso </td><td> BD Biosciences </td><td> BD FACSCalibur </td></tr></tbody></table>

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

La proteina chinasi C (PKC) famiglia di isoenzimi è coinvolta in numerosi processi fisiologici e patologici. I nostri dati recenti dimostrano che la PKC regola la funzione mitocondriale e lo stato di energia cellulare. Numerosi rapporti hanno dimostrato che l&#39;attivazione della PKC-a e PKC-ε migliora la funzione mitocondriale nel cuore ischemico e cardioprotettivo media. Al contrario, abbiamo dimostrato che PKC-α e ε-PKC sono coinvolte in nephrotoxicant indotta disfunzione mitocondriale e morte cellulare in cellule renali. Pertanto, l&#39;obiettivo di questo studio è stato quello di sviluppare un modello in vitro di cellule renali mantenere attive le funzioni mitocondriali in cui isoenzimi PKC potrebbe essere selettivamente attivati ​​o inibiti per determinare il loro ruolo nella regolazione della fosforilazione ossidativa e la sopravvivenza cellulare. Colture primarie di cellule renali del tubulo prossimale (RPTC) sono state coltivate in condizioni migliori con conseguente respirazione mitocondriale e l&#39;attività di mitomitocondriale enzimi simili a quelli in RPTC in vivo. Poiché le tecniche di transfezione tradizionali (Lipofectamine, elettroporazione) sono inefficienti in colture primarie e avere effetti negativi sulla funzione mitocondriale, PKC-ε cDNA mutanti sono state consegnate ai RPTC attraverso vettori adenovirali. Questo approccio risulta in trasfezione di oltre il 90% RPTC colta. Qui, vi presentiamo i metodi per valutare il ruolo della PKC-ε in: 1. regolazione della morfologia mitocondriale e funzioni associate alla sintesi di ATP, e 2. sopravvivenza della RPTC in coltura primaria. PKC-ε viene attivato overesprimono la costitutivamente attivo PKC-ε mutante. PKC-ε è inibita dalla sovraespressione della mutante inattivo di PKC-ε. Funzione mitocondriale viene valutata esaminando la respirazione, l&#39;integrità della catena respiratoria, attività dei complessi respiratori e F 0 F 1-ATPasi, tasso di produzione di ATP, e il contenuto di ATP. La respirazione è unssessed in digitonina permeabilizzate RPTC come stato 3 (respirazione massima in presenza di substrati in eccesso e ADP) e respirazioni disaccoppiati. Integrità della catena respiratoria è valutata misurando attività di tutti e quattro complessi della catena respiratoria nei mitocondri isolati. Capacità di fosforilazione ossidativa viene valutata misurando il potenziale di membrana mitocondriale, tasso di produzione di ATP, e l&#39;attività di F 0 F 1-ATPasi. Stato energetico del RPTC viene valutata determinando il contenuto intracellulare di ATP. Morfologia mitocondriale in cellule vive è visualizzato utilizzando MitoTracker Red 580, un colorante fluorescente che si accumula in particolare nei mitocondri, e monostrati vivi vengono esaminati al microscopio a fluorescenza. Vitalità RPTC è valutato in base annessina V / colorazione ioduro di propidio seguita mediante citometria di flusso per determinare apoptosi e oncosis. Questi metodi consentono di attivazione selettiva / inibizione della PKC individuale isoenzimiper valutare il loro ruolo in funzioni cellulari in una varietà di condizioni fisiologiche e patologiche che possono essere riprodotti in vitro.

La Figura 1 mostra che la consegna adenovirale di cDNA codificante le costitutivamente attivo (caPKC-ε) e inattivi (dnPKC-ε) mutanti di PKC-ε si traduce in livelli di proteine ​​significativamente aumentati di PKC-ε in RPTC e nei mitocondri. Le cellule infettate con cDNA che porta il caPKC-ε vettore sovraespresso il fosforilata (attiva) sotto forma di PKC-ε mentre le cellule infettate con cDNA codificante dnPKC-ε overexpressed PKC-ε che è stato inattivo (non fosforilata) (Figura 1)....

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Gli effetti di attivazione di proteina chinasi C (PKC) isoenzimi sulle funzioni mitocondriali associate respirazione e fosforilazione ossidativa e sulla vitalità cellulare sono descritti. L&#39;approccio si adatta tecnica adenovirale per sovraesprimere selettivamente isoenzimi PKC in coltura primaria e una varietà di saggi per determinare le funzioni mitocondriali e lo stato energetico della cellula.

La valutazione delle funzioni mitocondriali e la vitalità cellulare in cellule renali proteina chinasi C overexpressing isoenzimi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

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Biology

18.1K Views.  University of Arkansas for Medical Sciences. Gli effetti di attivazione di proteina chinasi C (PKC) isoenzimi sulle funzioni mitocondriali associate respirazione e fosforilazione ossidativa e sulla vitalità cellulare sono descritti. L'approccio si adatta tecnica adenovirale per sovraesprimere selettivamente isoenzimi PKC in coltura primaria e una varietà di saggi per determinare le funzioni mitocondriali e lo stato energetico della cellula.

Video: Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Conteggio e determinazione della vitalità delle cellule in coltura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Ricerca

Biologia

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Valutazione di espressione GFP e sostenibilità Utilizzando la Image-Based Tali Citometro

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D Imaging di aggregazione proteica in cellule vive

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

I saggi di redditività per le cellule in coltura

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Approcci sperimentale per lo studio mitocondriale localizzazione e funzione di un nucleare di cellule ciclo chinasi, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Colture primarie di cellule corticosurrenali ratto e saggi di Steroidogenic funzioni

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

La fotostimolazione da parte del laser Femtosecondo attiva la segnalazione o eventi mitocondriali regolati dal segnale extracellulare (ERK) nelle cellule bersaglio

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...