Förster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10.
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.
20 Related JoVE Articles!
Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain
Institutions: University of Michigan .
Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster
for genetic studies of cell cycle regulation in vivo
. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo
. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila
tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila
tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.
Molecular Biology, Issue 75, Cellular Biology, Developmental Biology, Anatomy, Physiology, Genetics, Flow Cytometry, Cell Cycle, DNA Replication, Metamorphosis, Biological, drosophila, Gal4/UAS, insect metamorphosis, animal model
Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area
Institutions: Louisiana State University Health Sciences Center.
Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5
. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8
. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo,
specific for each nuclei (Figure 1)
Neuroscience, Issue 66, Medicine, Physiology, midbrain, substantia nigra, ventral tegmental area, tyrosine hydroxylase, phosphorylation, nigrostriatal, mesoaccumbens, dopamine transporter
Identification of Post-translational Modifications of Plant Protein Complexes
Institutions: University of Warwick, Norwich Research Park, The Australian National University.
Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via
the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
Plant Biology, Issue 84, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
Imaging Through the Pupal Case of Drosophila melanogaster
Institutions: University of Miami.
The longstanding use of Drosophila
as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ
using live imaging. Most live imaging studies in Drosophila
have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila
have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ
. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo
may be generally useful in the study of cell and developmental biology in these tissues.
Basic Protocol, Issue 83, In vivo, live imaging, Drosophila, mitosis, wing, epithelium, metamorphosis, confocal microscopy
Imaging Centrosomes in Fly Testes
Institutions: University of Toledo.
Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila
spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila
is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila
testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.
Developmental Biology, Issue 79, biology (general), genetics (animal and plant), animal biology, animal models, Life Sciences (General), Centrosome, Spermatogenesis, Spermiogenesis, Drosophila, Centriole, Cilium, Mitosis, Meiosis
Live Imaging of Drosophila Larval Neuroblasts
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
Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis
Institutions: Concordia University.
This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans
embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans
is an excellent model organism to study tissue morphogenesis in vivo
due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.
Neuroscience, Issue 85, C. elegans, morphogenesis, cytokinesis, neuroblasts, anillin, microscopy, cell division
Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress
Institutions: CEA DSV iRCM SCSR, INSERM, U967, Université Paris Diderot, Sorbonne Paris Cité, Université Paris Sud, UMR 967.
Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.
Neuroscience, Issue 87, EdU, BrdU, in utero irradiation, neural stem and progenitor cells, cell cycle, embryonic cortex, immunostaining, cell cycle checkpoints, apoptosis, genotoxic stress, embronic mouse brain
Analysis of Cell Cycle Position in Mammalian Cells
Institutions: University of Western Ontario, University of Western Ontario.
The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1
offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5
. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.
Molecular Biology, Issue 59, cell cycle, proliferation, flow cytometry, DNA synthesis, fluorescence
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Institutions: Albert Einstein College of Medicine , Shanghai University of Traditional Chinese Medicine.
As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.
. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5
. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole's, binding residues on PXR6
. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or "gain-of-function" second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.
Biochemistry, Issue 81, Orphan nuclear receptor, ketoconazole, yeast two-hybrid, Pregnane X Receptor, ligand, antatogist, coactivators SRC-1 (steroid receptor coactivator 1), drug-receptor interaction
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Institutions: Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University.
Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1
. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2
). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4
, forming foci within 5-15 minutes5
. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6
. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9
. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6
. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11
. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12
. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
Genetics, Issue 67, Molecular Biology, Cellular Biology, Biochemistry, DNA, Double-strand breaks, DNA damage response, proteins, live cell imaging, 3D cell imaging, confocal microscopy
Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex
Institutions: Duke University Medical Center, Duke University Medical Center.
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.
Neuroscience, Issue 88, mitosis, radial glial cells, developing cortex, neural progenitors, brain slice, live imaging
Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos
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 (A
omplex or C
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.
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
Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells
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
Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations
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
). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8
). 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
). 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
). 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
Time-lapse Imaging of Mitosis After siRNA Transfection
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
Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
Institutions: AntiCancer, Inc..
There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.
Cellular Biology, Issue 13, Springer Protocols, hair follicles, liposomes, adenovirus, genes, stem cells
Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo
Institutions: University of California, Davis.
This protocol describes the use of the Drosophila melanogaster
syncytial embryo for studying mitosis1
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
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
Phase Contrast and Differential Interference Contrast (DIC) Microscopy
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Basic protocols, Issue 18, Current Protocols Wiley, Microscopy, Phase Contrast, Difference Interference Contrast