This work was supported by NIH grants CA133402, CA152313 and Department of Energy Office of Science DE-SC0001271. We thank the University of California Davis Flow Cytometry Shared Resource Laboratory with funding from the NCI P30 CA0933730, and NIH NCRR C06-RR12088, S10 RR12964 and S10 RR 026825 grants and with technical assistance from Ms. Bridget McLaughlin and Mr. Jonathan Van Dyke for their help with the flow cytometry experiments.

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The authors declare that they have no competing financial interests.

Like the proteins destined for other subcellular organelles, the mitochondrial targeted proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assistance of elaborate protein translocating and folding machines21,22. Mitochondria targeting sequences (MTS) obtained from exclusively mitochondrial resident proteins such as COX8 can be added to N-terminus of any gene sequence to target specific proteins into the mitochondria11,23,24. Here...

In mammals, cell cycle progression is dependent upon highly ordered events controlled by cyclins and cyclin-dependent kinases (Cdks)1. Through its cytoplasmic, nuclear, and centrosomal localization, CyclinB1/Cdk1 is able to synchronize different events in mitosis such as nuclear envelope breakdown and centrosome separation2. CyclinB1/Cdk1 protects mitotic cells against apoptosis3 and promotes mitochondrial fission, a critical step for an equal distribution of mitochondria to the newly formed daughter cells4.
In proliferating mammalian cells, mitochondrial ATP is generated via oxidative phosphorylation (OXPHOS) machinery (electron transport chain), which is composed of 5 multi-subunit complexes; complex I - complex V (CI-CV). Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase or complex I (CI) is the largest and least understood of the five complexes5. The complex consists of 45 subunits, 14 of which form the catalytic core. Once assembled, the complex assumes an L-shaped structure with one arm protruding into the matrix and the other arm embedded in the inner membrane6,7. Mutations in CI subunits are the cause of a variety of mitochondrial disorders8. A functionally efficient CI in OXPHOS is required not only for overall mitochondrial respiration9, but also for successful cell cycle progression10. Unravelling the mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease could enable the development of novel diagnostic procedures and advanced therapeutic strategies. In a recent study, we have found that the CyclinB1/Cdk1 complex translocates into mitochondria in the (Gap 2) G2/(Mitosis) M phase and phosphorylates CI subunits to enhance mitochondrial energy production, potentially to offset increased energy needs of cells during cell cycle11. Here we showcase experimental procedures and strategies that can be used to study mitochondrial translocation of otherwise nuclear/cytoplasmic kinases, their mitochondrial substrates as well as functional consequences of their mitochondrial localization using CyclinB1/Cdk1 as an example.
The finding that the CyclinB1/Cdk1 complex translocates into mitochondria when needed prompted the studies of mitochondria-specific overexpression and knockdown of this complex. To achieve mitochondria-specific expression of proteins, one can add a mitochondria targeting sequence (MTS) in the N-terminus of the protein of interest. Mitochondria targeting sequences allow the sorting of the mitochondrial proteins into the mitochondria where they normally reside12. We have used an 87 base mitochondria targeting sequence derived from the precursor of human cytochrome c oxidase subunit 8A (COX8) and cloned it into Green Fluorescent Protein (GFP)-tagged CyclinB1 or Red Fluorescent Protein (RFP)-tagged Cdk1 containing plasmids in frame. This method allowed us to target CyclinB1 and Cdk1 into the mitochondria, specifically changing the mitochondrial expression of these proteins without affecting their nuclear pool. By fluorescently tagging these proteins, we were able to monitor their localization in real time. Similarly, we have introduced MTS into a plasmid containing RFP-tagged dominant negative Cdk1, which allowed us to specifically knock down the mitochondrial expression and functions of Cdk1. It is essential to distinguish between mitochondrial and nuclear functions of the kinases that have dual localizations like Cdk1. Engineering MTS into the N-terminal of these dual functional kinases offers a great strategy that is easy to be employed and effective.
Since Cdk1 is a cell cycle kinase, it is fundamental to determine the cell cycle progression when Cdk1 is localized into mitochondria. To achieve this, we have utilized a new method to monitor DNA content in cells. Traditional methods include using BrdU (bromodeoxyuridine), a synthetic thymidine analogue, which incorporates into the newly synthesized DNA during the S phase of the cell cycle to substitute thymidine. Then the cells that are actively replicating their DNA can be detected using anti-BrdU antibodies. One disadvantage of this method is that it requires denaturation of DNA to provide access for the BrdU antibody by harsh methods like acid or heat treatment, which may result in inconsistency among results13,14. Alternatively, we utilized a similar approach to monitor the actively dividing cells with a different thymidine analog, EdU. EdU detection does not require harsh DNA denaturation as mild detergent treatment enables the detection reagent to access the EdU in newly synthesized DNA. The EdU method has proven to be more reliable, consistent and with potential for high-throughput analysis15.
Finally, to determine the mitochondrial substrates of Cdk1, we used a proteomics tool called 2D-DIGE, which is an advanced version of classical two-dimensional gel electrophoresis. Two dimensional electrophoresis separates proteins according to their isoelectric point in the first dimension and molecular weight in the second. Since post-translational modifications such as phosphorylation affect the isoelectric point and molecular weight of the proteins, 2D gels can detect the differences between phosphorylation statuses of proteins within different samples. The size (area and intensity) of protein spots changes with the expression level of proteins, allowing quantitative comparison between multiple samples. Using this method, we were able to differentiate the phosphorylated proteins in wild type versus mutant mitochondria-targeted Cdk1 expressing cells. The specific protein spots that showed in the wild type but were missing in the mitochondria-targeted mutant Cdk1 preparation were isolated and identified via mass spectrometry.
In traditional 2D gels, triphenylmethane dyes are used to visualize the proteins on the gel. 2D-DIGE uses fluorescent protein labels with minimal effect on protein electrophoretic mobility. Different protein samples can be labeled with different fluorescent dyes, mixed together and separated by the identical gels, allowing the co-electrophoresis of multiple samples on a single gel16. This minimizes the gel-to-gel variations, which is a critical problem in gel-based proteomics studies.

<table><tbody><tr><td>&lt;sup&gt;32&lt;/sup&gt;P ATP&amp;nbsp;</td><td>PerkinElmer</td><td>BLU002001MC&amp;nbsp;</td><td></td></tr><tr><td>Anti-mouse secondary antibody</td><td>Invitrogen&amp;nbsp;</td><td>A-11003</td><td>Alexa-546 conjugated</td></tr><tr><td>Anti-rabbit secondary antibody</td><td>Invitrogen&amp;nbsp;</td><td>A11029</td><td>Alexa-488 conjugated</td></tr><tr><td>ATP</td><td>Research Organics</td><td>1166A</td><td>For in vitro kinase assay</td></tr><tr><td>Cdk1 antibody</td><td>Cell Signaling Technology</td><td>9112</td><td></td></tr><tr><td>Cdk1 kinase buffer</td><td>New England Biolabs</td><td>P6020S</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Life Technologies</td><td>C10337</td><td>For cell cycle analysis with EdU labeling</td></tr><tr><td>COX IV antibody</td><td>Cell Signaling Technology</td><td>4844S</td><td>For mitochondrial immunostaining</td></tr><tr><td>Cyclin B1 antibody</td><td>Santa Cruz Biotech</td><td>sc-752</td><td></td></tr><tr><td>CyclinB1/Cdk1 enzyme complex</td><td>New England Biolabs</td><td>P6020S</td><td>Avoid freeze/thaw</td></tr><tr><td>CyDye DIGE Fluor Labeling Kit</td><td>GE Healthcare Life Sciences</td><td>25-8009-83</td><td></td></tr><tr><td>DIGE Gel and DIGE Buffer Kit</td><td>GE Healthcare Life Sciences</td><td>28-9480-26 AA</td><td></td></tr><tr><td>Dimethylformamide&amp;nbsp;</td><td>Sigma Aldrich</td><td>319937</td><td>DMF</td></tr><tr><td>Dithiothreitol</td><td>Bio-Rad</td><td>161-0611</td><td>DTT</td></tr><tr><td>dNTP</td><td>EMD Millipore</td><td>71004</td><td>For site-directed mutagenesis</td></tr><tr><td>Dpn I enzyme</td><td>Stratagene</td><td>200519-53</td><td>For site-directed mutagenesis</td></tr><tr><td>Dry Strip cover fluid</td><td>GE Healthcare Life Sciences</td><td>17-1335-01</td><td>Used as mineral oil</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>4040-03</td><td></td></tr><tr><td>EGTA</td><td>Acros Organics</td><td>409910250</td><td></td></tr><tr><td>Eppendorf Vacufuge Concentrator</td><td>Fisher Scientific</td><td>07-748-13</td><td>Used as vacuum centrifuge concentrator</td></tr><tr><td>Fluoromount G</td><td>Southern Biotech</td><td>0100-01</td><td>Anti-fade mounting solution</td></tr><tr><td>Fortessa Flow Cytometer</td><td>BD Biosciences</td><td>649908</td><td>For cell cycle analysis with EdU labeling</td></tr><tr><td>Histone H1</td><td>Calbiochem</td><td>382150</td><td>For in vitro kinase assay</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>For purifying DNA fragments from agarose gels</td></tr><tr><td>Immobiline DryStrip Gels</td><td>GE Healthcare Life Sciences</td><td>18-1016-61</td><td>IEF (isoelectric focusing) strips</td></tr><tr><td>Immobilized Glutathione</td><td>Thermo Scientific</td><td>15160</td><td>Glutathione-agarose beads</td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich</td><td>I1149</td><td>IAA</td></tr><tr><td>IPGphor 3 Isoelectric Focusing Unit</td><td>GE Healthcare Life Sciences</td><td>11-0033-64</td><td>IPGphor strip holders</td></tr><tr><td>Isopropyl-b-D-thio-galactopyranoside&amp;nbsp;</td><td>RPI Corp</td><td>156000-5.0</td><td>IPTG</td></tr><tr><td>Leupeptin</td><td>Sigma Aldrich</td><td>L9783</td><td>For cell lysis buffer</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668027</td><td>Transfection reagent</td></tr><tr><td>Lysine</td><td>Sigma Aldrich</td><td>L5501</td><td>For CyDye labeling</td></tr><tr><td>Lysozyme</td><td>EMD Chemicals</td><td>5960</td><td></td></tr><tr><td>Mitoctracker Red/Green</td><td>Invitrogen&amp;nbsp;</td><td>M7512/M7514</td><td>Mitochondrial fluorescent dyes</td></tr><tr><td>MOPS</td><td>EMD Chemicals</td><td>6310</td><td></td></tr><tr><td>pEGFP-N1</td><td>Clonetech</td><td>6085-1</td><td>GFP-expressing vector</td></tr><tr><td>Pfu</td><td>Stratagene</td><td>600-255-52</td><td></td></tr><tr><td>pGEX-5X-1&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>28-9545-53</td><td>GST-expressing vector</td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Shelton Scientific</td><td>IB01090</td><td>PMSF</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>14040</td><td>PBS</td></tr><tr><td>Spectra/Por 4 dialysis tubing</td><td>Spectrum Labs</td><td>132700</td><td>as porous membrane tubing for dialysis</td></tr><tr><td>Pro-Q Diamond Phosphoprotein Gel Stain</td><td>Life Technologies</td><td>P-33300</td><td>For staining phosphoproteins on 2D gels</td></tr><tr><td>Proteinase inhibitor cocktail</td><td>Calbiochem</td><td>539134</td><td>For cell lysis buffer</td></tr><tr><td>QuikChange site-directed mutagenesis kit</td><td>Stratagene</td><td>200519-5</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td>MiniPrep Plasmid Isolation Kit</td></tr><tr><td>RO-3306</td><td>Alexis Biochemicals</td><td>270-463-M001</td><td>Cdk1 inhibitor</td></tr><tr><td>Rotenone</td><td>MP Biomedicals</td><td>150154</td><td>Complex I inhibitor</td></tr><tr><td>Sodium carbonate</td><td>Fisher Scientific</td><td>S93359</td><td></td></tr><tr><td>Sodium chloride</td><td>EMD Chemicals</td><td>SX0420-5</td><td>For cell lysis buffer</td></tr><tr><td>Sodium orthovanadate</td><td>MP Biomedicals</td><td>159664</td><td>For cell lysis buffer</td></tr><tr><td>Sodium pyrophosphate decahydrate</td><td>Alfa Aesar</td><td>33385</td><td>For cell lysis buffer</td></tr><tr><td>Sodium &amp;beta;-glycerophosphate</td><td>Alfa Aesar</td><td>L03425</td><td>For cell lysis buffer</td></tr><tr><td>SpectraMax M2e&amp;nbsp;</td><td>Molecular Devices</td><td>M2E</td><td>Microplate reader</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>57-50-1</td><td></td></tr><tr><td>Tissue Grinder pestle</td><td>Kimble Chase</td><td>885301-0007</td><td>For mitochondria isolation</td></tr><tr><td>Tissue Grinder tube</td><td>Kimble Chase</td><td>885303-0007</td><td>For mitochondria isolation</td></tr><tr><td>Trichloroacetic acid solution</td><td>Sigma Aldrich</td><td>T0699</td><td>TCA</td></tr><tr><td>Tris</td><td>MP Biomedicals</td><td>103133</td><td></td></tr><tr><td>Triton-x-100</td><td>Teknova</td><td>T1105</td><td></td></tr><tr><td>Trypsin</td><td>Calbiochem</td><td>650211</td><td></td></tr><tr><td>Typhoon Imager</td><td>GE Healthcare Life Sciences</td><td>28-9558-09</td><td>Laser gel scanner fro 2D-DIGE</td></tr><tr><td>Ubiquinone</td><td>Sigma Aldrich</td><td>C7956</td><td></td></tr></tbody></table>

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.

Sub-mitochondrial localization of CyclinB1 and Cdk1
Sodium carbonate extraction is used to determine whether a protein is located inside the mitochondria or on the outside surface, namely outer membrane. Once a protein is shown to localize inside the mitochondria, further determination of sub-mitochondrial localization can be made via mitoplasting combined with protease digestion. To specify the sub-mitochondria...

Watch this Scientific Journal Video about Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1 at JoVE.com

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

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.

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

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11.8K Views.  University of California, Davis. 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. 

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

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Biochemistry

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

Neuroscience

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

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.

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

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

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

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