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Pubmed Article
Mitotic spindle proteomics in Chinese hamster ovary cells.
PUBLISHED: 03-30-2011
Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.
Authors: Louis-Jan Pilaz, Debra L. Silver.
Published: 06-04-2014
Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.
18 Related JoVE Articles!
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Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
Authors: Dominik Schnerch, Marie Follo, Julia Felthaus, Monika Engelhardt, Ralph Wäsch.
Institutions: University Medical Center Freiburg.
Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1. Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5. To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6. To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.
Genetics, Issue 67, Cellular Biology, Molecular Biology, Proteomics, Cyclin B, spindle assembly checkpoint, anaphase-promoting complex, mitosis, proteasome-dependent proteolysis, SNAP, cell cycle
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Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs
Authors: Judith A. Sharp, Mike D. Blower.
Institutions: Massachusetts General Hospital, Harvard Medical School.
Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.
Molecular Biology, Issue 76, Genetics, Developmental Biology, Biochemistry, Bioengineering, Cellular Biology, RNA, Messenger, Stored, RNA Processing, Post-Transcriptional, Xenopus, microtubules, egg extract, purification, RNA localization, mRNA, Xenopus tropicalis, eggs, animal model
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Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae
Authors: Diana Zitserman, Fabrice Roegiers.
Institutions: Fox Chase Cancer Center.
Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway. Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).
Neuroscience, Issue 51, Live imaging, asymmetric cell division, Drosophila, pupa
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Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes
Authors: Asa Hagner-McWhirter, Maria Winkvist, Stephanie Bourin, Rita Marouga.
Institutions: GE Healthcare Bio-Sciences AB.
Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.
Biochemistry, Issue 21, Cell surface protein labelling, Ettan DIGE, CyDye DIGE Fluor minimal dyes, cell surface proteins, receptors, fluorescence, 2-D electrophoresis
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Nuclear Transfer into Mouse Oocytes
Authors: Dieter Egli, Kevin Eggan.
Institutions: Harvard.
Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).
Developmental Biology, Issue 1, oocytes, nuclear transfer, stem cells
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FIBS-enabled Noninvasive Metabolic Profiling
Authors: Alireza Behjousiar, Antony Constantinou, Karen M. Polizzi, Cleo Kontoravdi.
Institutions: Imperial College London, Imperial College London.
In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on Förster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.
Bioengineering, Issue 84, metabolite monitoring, in vivo biosensors, in situ monitoring, mammalian cell culture, bioprocess engineering, medium formulation
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Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System
Authors: Oren Levy, Priya Anandakumaran, Jessica Ngai, Rohit Karnik, Jeffrey M. Karp.
Institutions: Brigham and Women's Hospital, Brigham and Women's Hospital, Harvard University, Harvard University, Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology.
A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.
Bioengineering, Issue 80, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
Authors: Titiwat Sungkaworn, Finn Rieken, Martin J. Lohse, Davide Calebiro.
Institutions: University of Würzburg, Germany.
Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., β2-adrenergic and γ-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.
Bioengineering, Issue 89, pharmacology, microscopy, receptor, live-cell imaging, single-molecule, total internal reflection fluorescence, tracking, dimerization, protein-protein interactions
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Time-lapse Imaging of Mitosis After siRNA Transfection
Authors: Douglas R. Mackay, Katharine S. Ullman, Christopher K. Rodesch.
Institutions: University of Utah, University of Utah.
Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.
Cellular Biology, Issue 40, microscopy, live imaging, mitosis, transfection, siRNA
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Quantitative Analysis of Chromatin Proteomes in Disease
Authors: Emma Monte, Haodong Chen, Maria Kolmakova, Michelle Parvatiyar, Thomas M. Vondriska, Sarah Franklin.
Institutions: David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah.
In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options. Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.
Medicine, Issue 70, Molecular Biology, Immunology, Genetics, Genomics, Physiology, Protein, DNA, Chromatin, cardiovascular disease, proteomics, mass spectrometry
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Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes
Authors: Poojitha Sitaram, Sarah Grace Hainline, Laura Anne Lee.
Institutions: Vanderbilt University Medical Center.
Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.
Basic Protocol, Issue 83, Drosophila melanogaster, dissection, testes, spermatogenesis, meiosis, germ cells, phase-contrast microscopy, immunofluorescence
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The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Authors: Monica Soldi, Tiziana Bonaldi.
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
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Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
Authors: Jaana Mannik, Alex Meyers, Paul Dalhaimer.
Institutions: University of Tennessee, University of Tennessee.
Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques. In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins. In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions. The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.
Bioengineering, Issue 86, Lipid droplet, lipid body, fat body, oil body, Yeast, placenta, placental villous cells, isolation, purification, density gradient centrifugation
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Live Imaging of Drosophila Larval Neuroblasts
Authors: Dorothy A. Lerit, Karen M. Plevock, Nasser M. Rusan.
Institutions: National Institutes of Health.
Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.
Neuroscience, Issue 89, live imaging, Drosophila, neuroblast, stem cell, asymmetric division, centrosome, brain, cell cycle, mitosis
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Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos
Authors: Maureen Sinclair, Jun-Yong Huang.
Institutions: University of Newcastle, United Kingdom.
The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive. Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9. Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.
Cellular Biology, Issue 64, Developmental Biology, Spindle assembly checkpoint (SAC), Mitosis, Laser scanning confocal microscopy system, Kinetochore, Drosophila melanogaster, Syncytial embryo
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Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells
Authors: Asher Castiel, Leonid Visochek, Leonid Mittelman, Yael Zilberstein, Francoise Dantzer, Shai Izraeli, Malka Cohen-Armon.
Institutions: Sheba Medical Center, Tel-Aviv University, Tel-Aviv University, Tel-Aviv University, Ecole Superieure de Biotechnologie Strasbourg, Tel-Aviv University.
Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.
Cancer Biology, Issue 78, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Genetics, Neoplastic Processes, Pharmacologic Actions, Live confocal imaging, Extra-centrosomes clustering/de-clustering, Mitotic Catastrophe cell death, PJ-34, myocardial infarction, microscopy, imaging
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Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology
Authors: Matthew Dickerson, Kristen Leong, Kate Sheldon, Lara Madison.
Institutions: BioScale, Inc., BioScale, Inc..
Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays. An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass. To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate. Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.
Bioengineering, Issue 48, Immunoassays, Chinese Hamster Ovary Host Cell Protein, Residual Protein A assay, Assay development, Biomarker detection and quantitation, Phospho-AKT, Gadd34, tissue sample, tumor sample, bioreactor sample
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Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo
Authors: Ingrid Brust-Mascher, Jonathan M. Scholey.
Institutions: University of California, Davis.
This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, ≈10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.
Developmental Biology, Issue 31, mitosis, Drosophila melanogaster syncytial embryo, microinjection, protein inhibition
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