Ce travail a été soutenu par une subvention de la National Institutes of Health, l&#39;Institut national du diabète et des maladies digestives et rénales, 2R01DK59558 (à GN). UAMS Translational Research Institute soutenu par le National Institutes of Health Centre national de recherche sur les ressources de subvention UL1 RR029884 fourni un financement partiel pour le Core cytométrie en flux à UAMS. Nous remercions le Dr Peipei Ping (Université de Californie à Los Angeles, Los Angeles, CA) pour fournir une aliquote de l&#39;adénovirus portant l&#39;ADNc codant dominant négatif (inactif) mutant de la PKC-ε et le Dr Allen Samarel (Loyola University Medical Center; Maywood, IL) pour fournir un aliquot de vecteur adénoviral codant le mutant constitutivement active de la PKC-ε. Nous remercions également MM. Peter Parker et Peter Sugden (Imperial College London, Londres, Royaume-Uni) pour fournir des ADNc codant constitutivement actives 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. 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Aucun conflit d&#39;intérêt déclaré.

L&#39;approche présentée ici permet la surexpression des isozymes de la PKC individuelles dans la culture primaire de cellules tubulaires proximales rénales. Il ya plusieurs points forts de cette approche: 1. Il permet d&#39;enquêter sur les mécanismes de régulation dans une population homogène de cellules (cellules tubulaires proximales rénales) qui sont la principale cible des insultes diverses (ischémie, l&#39;hypoxie, stress oxydatif), les médicaments et nephrotoxicants dans le rein. 2. Fonctions mitochond...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Hotte à flux laminaire </td><td> Thermo Electron Corporation </td><td> FORMA 1104 </td></tr><tr><td> 2 ml et 15 ml broyeur Dounce tissus </td><td> WHEATON </td><td> 989-24607, 357544 </td></tr><tr><td> 85 et 235 microns maille de nylon </td><td> Petites pièces </td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> Tube 50 ml à centrifuger stérile </td><td> Biologix </td><td> BCT-P 50BS </td></tr><tr><td> 1,5 ml du tube micro </td><td> Sarstedt </td><td> 72.690.001 </td></tr><tr><td> 35 x 10 mm plats de culture stériles </td><td> Corning </td><td> 430165 </td></tr><tr><td> Centrifugeuse Jouan </td><td> Jouan </td><td> Jouan CR3 11175704 Rotors: Jouan T40 </td></tr><tr&gt; <td> Réglable micro-centrifugeuse </td><td> SIGMA </td><td> Modèle 1 à 15 </td></tr><tr><td> Moniteur d&#39;oxygène biologique </td><td> YSI Incorporated </td><td> YSI Modèle 5300A </td></tr><tr><td> Simple système d&#39;oxygène Chambre Micro </td><td> YSI Incorporated </td><td> 5356S </td></tr><tr><td> Sonde d&#39;oxygène </td><td> YSI Incorporated </td><td> 5331A </td></tr><tr><td> Bain à circulation </td><td> YSI Incorporated </td><td> 5310 </td></tr><tr><td> Kit de KCl et normalisée de la membrane </td><td> YSI Incorporated </td><td> 5775 </td></tr><tr><td> Agitateur magnétique </td><td> YSI Incorporated </td><td> 5222 </td></tr><tr><td> Enregistreur à plat </td><td> Kipp &amp; Zonen </td><td> BD 11E </td></tr><tr><td> 48 puits et 96 puits ont été transparentes </td><td> Costar </td><td> 3548, 3679 </td></tr><tr><td> Thermomixer R </td><td> Eppendorf </td><td> 5355 21919 </td></tr><tr><td> Agitateur orbital MAXQ 2000 </td><td> Thermo Scientific </td><td> Shka 2000 </td></tr><tr><td> Spectra Fluor Plus (absorbance / fluorescence / luminescence lecteur) </td><td> Tecan </td><td> F129005 </td></tr><tr><td> Chemise d&#39;eau américain Autoflow automatique CO 2 incubateur </td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td> 12x75 mm tubes en polystyrène de culture d&#39;essai pour la cytométrie en flux </td><td> Marque Fisher </td><td> 14-961-20 </td></tr><tr><td> Axioskop Objectif à immersion d&#39;eau 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 à 830 - PB </td></tr><tr><td> DMEM / Ham F12 </td><td> Sigma </td><td> D 2906 - 1L </td></tr><tr><td> Mésylate de déféroxamine </td><td> Hospira </td><td> D110 </td></tr><tr><td> Collagénase de type I </td><td> Worthington </td><td> 4196 </td></tr><tr><td> Inhibiteur de la trypsine </td><td> Sigma </td><td> T 6522 - 500mg </td></tr><tr><td> 5,5 &#39;, 6,6&#39;-tétrachloro-1, 1 &#39;, 3,3&#39;-tetraethylbenzimidazolylcarbocyanine iodure (JC-1) </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> Mitotracker Rouge </td><td> Invitrogen </td><td> M22425 </td></tr><tr><td> ATP bioluminescence Assay Kit HS II </td><td> Roche </td><td> 11 699 709 001 </td></tr><tr><td> Annexine V - solution de FITC </td><td> BioVision </td><td> 1001 - 200 </td></tr><tr><td> Cytomètre en flux </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 protéine kinase C (PKC) de la famille des isoenzymes est impliqué dans de nombreux processus physiologiques et pathologiques. Nos données récentes démontrent que la PKC régule la fonction mitochondriale et l&#39;état énergétique cellulaire. De nombreux rapports ont démontré que l&#39;activation de la PKC-a et de la PKC-ε améliore la fonction mitochondriale dans la cardiopathie ischémique et cardioprotection médiateur. En revanche, nous avons montré que la PKC-α et PKC-ε sont impliqués dans nephrotoxicant induite par la dysfonction mitochondriale et la mort cellulaire dans les cellules rénales. Par conséquent, l&#39;objectif de cette étude était de développer un modèle in vitro de cellules rénales maintenir actives les fonctions mitochondriales dans lequel isoenzymes de PKC pourrait être sélectivement activés ou inhibés afin de déterminer leur rôle dans la régulation de la phosphorylation oxydative et la survie cellulaire. Les cultures primaires de cellules tubulaires proximales rénales (RPTC) ont été cultivées dans de meilleures conditions résultant de la respiration mitochondriale et l&#39;activité des mitochondrial enzymes analogues à celles RPTC in vivo. Parce que les techniques de transfection traditionnels (Lipofectamine, l&#39;électroporation) sont inefficaces dans des cultures primaires et avoir des effets néfastes sur la fonction mitochondriale, PKC-ε ADNc mutants ont été livrés à travers RPTC vecteurs adénoviraux. Cette approche se traduit par la transfection de plus de 90% RPTC culture. Ici, nous présentons les méthodes pour évaluer le rôle de la PKC-ε dans: 1. la régulation de la morphologie des mitochondries et des fonctions associées à la synthèse d&#39;ATP, et 2. survie des RPTC en culture primaire. PKC-ε est activé par surexprimant constitutivement active de la PKC ε-mutant. PKC-ε est inhibée par la surexpression de la mutant inactif de la PKC-ε. La fonction mitochondriale est évaluée en examinant la respiration, de l&#39;intégrité de la chaîne respiratoire, les activités des complexes respiratoires et F 0 F 1-ATPase, le taux de production d&#39;ATP, et la teneur en ATP. La respiration est unssessed dans digitonine-perméabilisées RPTC que l&#39;état 3 (respiration maximale en présence de substrats en excès et ADP) et les respirations découplés. L&#39;intégrité de la chaîne respiratoire est évaluée par la mesure des activités des quatre complexes de la chaîne respiratoire dans les mitochondries isolées. Capacité de phosphorylation oxydative est évaluée par mesure du potentiel de membrane mitochondriale, le taux de production d&#39;ATP, et une activité de F 0 F 1-ATPase. Statut énergétique de RPTC est évaluée par la détermination de la teneur en ATP intracellulaire. Morphologie mitochondriale dans les cellules vivantes est visualisée à l&#39;aide Mitotracker Red 580, un colorant fluorescent qui s&#39;accumule spécifiquement dans les mitochondries, et des monocouches vivants sont examinés sous un microscope à fluorescence. RPTC viabilité est évaluée à l&#39;aide annexine V / iodure de propidium suivie coloration par cytométrie en flux afin de déterminer l&#39;apoptose et oncose. Ces méthodes permettent une activation sélective / inhibition de la PKC individuelle isozymesafin d&#39;évaluer leur rôle dans les fonctions cellulaires dans une variété de conditions physiologiques et pathologiques qui peuvent être reproduits in vitro.

La figure 1 montre que la livraison adénoviral de l&#39;ADNc codant les constitutivement actives (caPKC-ε) et inactifs (dnPKC-ε) des mutants de la PKC-ε aboutit à des niveaux beaucoup plus élevés de la protéine PKC-ε dans RPTC et dans les mitochondries. Les cellules infectées avec le vecteur portant l&#39;ADNc caPKC-ε surexprimé la phosphorylé (actif) sous forme de PKC-ε alors que les cellules infectées avec l&#39;ADNc codant dnPKC-ε-ε surexprimé PKC qui est inactif (pas phosphorylée)...

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

Les effets de l&#39;activation de la protéine kinase C (PKC) isozymes de la fonction mitochondriale associés à la respiration et la phosphorylation oxydative et sur la viabilité cellulaire sont décrites. L&#39;approche technique s&#39;adapte adénoviral de façon sélective surexprimer isoenzymes de PKC dans une culture cellulaire primaire et une variété de tests pour déterminer les fonctions mitochondriales et l&#39;état énergétique de la cellule.

Évaluation des fonctions mitochondriales et la viabilité cellulaire dans les cellules rénales surexprimant la protéine kinase C Isozymes

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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18.1K vues.  University of Arkansas for Medical Sciences. Les effets de l'activation de la protéine kinase C (PKC) isozymes de la fonction mitochondriale associés à la respiration et la phosphorylation oxydative et sur la viabilité cellulaire sont décrites. L'approche technique s'adapte adénoviral de façon sélective surexprimer isoenzymes de PKC dans une culture cellulaire primaire et une variété de tests pour déterminer les fonctions mitochondriales et l'état énergétique de la cellule.

Vidéo: 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.

Comptage et détermination de la viabilité des cellules cultivées

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

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Biologie

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.

L&#39;activation de l&#39;apoptose par micro-injection cytoplasmique du cytochrome 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.

Evaluation de l&#39;expression de GFP et viabilité en utilisant le Tali Image Based Cytomètre

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 imagerie de l&#39;agrégation des protéines dans les cellules vivantes

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.

Les analyses de viabilité de cellules en culture

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.

Approches expérimentales pour l&#39;étude mitochondrial Localisation et fonction d&#39;une cellule du cycle nucléaire Kinase, 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.

Mesure simultanée de mitochondrial Calcium et mitochondrial potentiel membranaire dans des cellules vivantes par microscopie à fluorescence

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.

Une interface utilisateur graphique pour le suivi assisté par logiciel de la concentration de protéines dans les protrusions cellulaires dynamiques

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.

Culture primaire de cellules Corticosurrénaliennes Rat et tests des fonctions Stéroïdogéniques

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.

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

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