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Pubmed Article
H-Ras mediates the inhibitory effect of epidermal growth factor on the epithelial Na+ channel.
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PLoS ONE
PUBLISHED: 03-17-2015
The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.
Authors: Melanie R. Rutkowski, Michael J. Allegrezza, Nikolaos Svoronos, Amelia J. Tesone, Tom L. Stephen, Alfredo Perales-Puchalt, Jenny Nguyen, Paul J. Zhang, Steven N. Fiering, Julia Tchou, Jose R. Conejo-Garcia.
Published: 03-26-2014
ABSTRACT
Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.
22 Related JoVE Articles!
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A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide
Authors: Mulu Geletu, Stephanie Guy, Kevin Firth, Leda Raptis.
Institutions: Queen's University, Ask Science Products Inc..
In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.
Molecular Biology, Issue 92, Electroporation, Indium-Tin oxide, signal transduction, gap junctional communication, peptides, Stat3
51710
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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Authors: Lori E. Lowes, Benjamin D. Hedley, Michael Keeney, Alison L. Allan.
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
51248
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A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion
Authors: Archis Bagati, Zethan Koch, Diane Bofinger, Haneesha Goli, Laura S. Weiss, Rosie Dau, Megha Thomas, Shoshanna N. Zucker.
Institutions: Roswell Park Cancer Institute, D'Youville College.
Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted “X”). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.
Medicine, Issue 98, hormone, cytokine, growth factor, migration, invasion, collagen, cancer
51480
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Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
Authors: Matteus Krappitz, Christoph Korbmacher, Silke Haerteis.
Institutions: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU).
The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
Biochemistry, Issue 89, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments
51582
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
51763
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Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres
Authors: Virginia Espina, Kirsten H. Edmiston, Lance A. Liotta.
Institutions: George Mason University, Virginia Surgery Associates.
Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue. Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.
Cancer Biology, Issue 93, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
51926
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Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Authors: Nadine Rommerswinkel, Bernd Niggemann, Silvia Keil, Kurt S. Zänker, Thomas Dittmar.
Institutions: Witten/Herdecke University.
The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.
Bioengineering, Issue 92, cell migration, 3D collagen matrix, cell tracking
51963
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Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans
Authors: Carmen I. Nussbaum-Krammer, Mário F. Neto, Renée M. Brielmann, Jesper S. Pedersen, Richard I. Morimoto.
Institutions: Northwestern University.
Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.
Cellular Biology, Issue 95, Caenorhabditis elegans, neurodegenerative diseases, protein misfolding diseases, prion-like spreading, cell-to-cell transmission, protein aggregation, non-cell autonomous toxicity, proteostasis
52321
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Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow
Authors: Hafsa Munir, G. Ed Rainger, Gerard B. Nash, Helen McGettrick.
Institutions: University of Birmingham, University of Birmingham, University of Birmingham.
Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.
Immunology, Issue 95, Endothelial cells, leukocytes, mesenchymal stromal cells, mesenchymal stem cells, co-culture, adhesion, inflammation, recruitment, flow based adhesion assay, Ibidi microslide, neutrophil
52480
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Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis
Authors: Denise Wernike, Chloe van Oostende, Alisa Piekny.
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
51188
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The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF
Authors: Wen Xiong, Yan Gao, Xun Cheng, Charles Martin, Dongmei Wu, Shuyuan Yao, Min-Ju Kim, Yang Liu.
Institutions: Stemgent, Stemgent.
Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.
Cellular Biology, Issue 33, SC1(Pluripotin), LIF, mESC, mouse ESC, mouse ES cells, pluripotency, self-renewal, small molecule
1550
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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Authors: Christophe. J Queval, Ok-Ryul Song, Vincent Delorme, Raffaella Iantomasi, Romain Veyron-Churlet, Nathalie Deboosère, Valérie Landry, Alain Baulard, Priscille Brodin.
Institutions: Université de Lille.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.
Infection, Issue 83, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
51114
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Primary Human Bronchial Epithelial Cells Grown from Explants
Authors: Asma Yaghi, Aisha Zaman, Myrna Dolovich.
Institutions: McMaster University.
Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.
Medicine, Issue 37, Human bronchus, epithelium, primary culture, permeable support, cilia
1789
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Bimolecular Fluorescence Complementation
Authors: Katy A. Wong, John P. O'Bryan.
Institutions: University of Illinois at Chicago.
Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.
Cellular Biology, Issue 50, Fluorescence, imaging, compartmentalized signaling, subcellular localization, signal transduction
2643
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Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Authors: Xuehua Xu, Tian Jin.
Institutions: National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits 7, 9, 10. Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.
Molecular Biology, Issue 55, Chemotaxis, directional sensing, GPCR, PCR, G-proteins, signal transduction, Dictyostelium discoideum
3128
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Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts
Authors: Urszula M. Polanska, Ahmet Acar, Akira Orimo.
Institutions: University of Manchester, Juntendo University.
Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3. The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11. We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.
Medicine, Issue 56, cancer, stromal myofibroblasts, experimentally generated carcinoma-associated fibroblasts (exp-CAFs), fibroblast, human mammary carcinomas, tumour xenografts
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Spheroid Assay to Measure TGF-β-induced Invasion
Authors: Hildegonda P.H. Naber, Eliza Wiercinska, Peter ten Dijke, Theo van Laar.
Institutions: Leiden University Medical Centre.
TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6. To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.
Medicine, Issue 57, TGF-β, TGF, breast cancer, assay, invasion, collagen, spheroids, oncology
3337
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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Authors: Grażyna Nowak, Diana Bakajsova.
Institutions: University of Arkansas for Medical Sciences .
The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC. Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis. These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.
Cellular Biology, Issue 71, Biochemistry, Molecular Biology, Genetics, Pharmacology, Physiology, Medicine, Protein, Mitochondrial dysfunction, mitochondria, protein kinase C, renal proximal tubular cells, reactive oxygen species, oxygen consumption, electron transport chain, respiratory complexes, ATP, adenovirus, primary culture, ischemia, cells, flow cytometry
4301
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Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Authors: Catheleyne D'hondt, Bernard Himpens, Geert Bultynck.
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
50443
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A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster
Authors: Ajay Srivastava.
Institutions: Western Kentucky University .
Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.
Medicine, Issue 79, Imaginal Discs, Drosophila melanogaster, Neoplasm Metastasis, Drosophila, Invasive Tumors, Benign Tumors, Cephalic Complex, Mosaic Analysis with a Repressible Cell Marker technique
50624
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A Method for Culturing Embryonic C. elegans Cells
Authors: Rachele Sangaletti, Laura Bianchi.
Institutions: University of Miami .
C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.
Developmental Biology, Issue 79, Eukaryota, Biological Phenomena, Cell Physiological Phenomena, C. elegans, cell culture, embryonic cells
50649
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Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model
Authors: Nicola H. Green, Bernard M. Corfe, Jonathan P. Bury, Sheila MacNeil.
Institutions: University of Sheffield, University of Sheffield, Sheffield Teaching Hospitals NHS Foundation Trust.
The incidence of both esophageal adenocarcinoma and its precursor, Barrett’s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa. A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa. This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett’s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.
Bioengineering, Issue 99, esophagus, epithelium, tissue engineering, 3D construct, esophageal cancer, Barrett’s Metaplasia
52693
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