Imaging the division process in living tissue culture cells

Khodjakov, Alexey; Rieder, Conly L.

doi:10.1016/j.ymeth.2005.07.007

Cited by 76 publications

(58 citation statements)

References 61 publications

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“…Wounds were made with the tip of a micropipette or by removing half of the monolayer with a sterile razor blade. Cells were maintained in modified Rose chambers (29) in Leibovitz's L-15 medium without phenol red (Invitrogen) with 10% FBS and antibiotics. To analyze cell motility, phase contrast time-lapse microscopy was done in a 37jC room using Axiovert 200M (Zeiss) microscope equipped with a 10Â/ 0.25 NA CP-Achromat objective and AxioCam MR camera.…”

Section: Methodsmentioning

confidence: 99%

Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells

et al. 2006

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The first steps of invasion and metastasis include the dissociation of adherens junctions and the induction of migratory phenotype, through a program that resembles epithelial-mesenchymal transition (EMT). The L1 cell adhesion molecule, which is normally found primarily in the brain, was recently shown to be expressed in different types of cancer and to have tumor-promoting activity. We now find that L1 mediates EMT-like events in MCF7 breast carcinoma cells. MCF7 predominantly expresses the nonneuronal isoform of L1, as do 16 of 17 other cell lines derived from different types of cancer. L1 protein expression in MCF7 cells, which form E-cadherin-containing adherens junctions, is inversely related to cell density. Analysis of MCF7 cells with overexpression or knockdown of nonneuronal L1 isoform revealed that L1 expression leads to the disruption of adherens junctions and increases B-catenin transcriptional activity. As a result, L1 expression promotes the scattering of epithelial cells from compact colonies. Expression of the fulllength L1 protein, but not of its soluble extracellular moiety, increases the motility of the MCF7 epithelial monolayer in a wound-healing assay, in which L1 expression is preferentially observed and required in cells leading the movement of the monolayer. Based on these results, we propose a model for the role of L1 as a trigger of EMT-like events in transformed epithelial cells.

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Section: Methodsmentioning

confidence: 99%

Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells

et al. 2006

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“…Coverslip cultures were subsequently mounted in Rose chambers (Khodjakov and Rieder 2006) filled with Schneider's medium and 10% FBS. For time-lapse recordings, cells were visualized by DIC as they entered mitosis, and images were collected every 30 s on the DeltaVision microscope described previously.…”

Section: Cells and Immunofluorescencementioning

confidence: 99%

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

et al. 2006

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Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40-50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4-6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly,

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“…With recent advances in imaging and fluorescent protein technology, live imaging has become a routine aspect of cellular analysis 2 . A variety of GFP (and other color variants 3 ) -tagged proteins are widely available or relatively easy to construct and can be used to track a number of cell division hallmarks including DNA/chromosome dynamics 4,5 , centrosome duplication 6 , cyclin B dynamics 7 , nuclear envelope breakdown and reassembly 8,9 , mitotic spindle formation 10 , and various stages of cytokinesis 11 .…”

Section: Discussionmentioning

confidence: 99%

Time-lapse Imaging of Mitosis After siRNA Transfection

Mackay

Ullman

Rodesch

2010

JoVE

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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. Video LinkThe video component of this article can be found at https://www.jove.com/video/1878/ Protocol siRNA transfection and cell preparation 1. Prepare a 4-well LabTek II chambered coverglass by adding 0.5mL of fibronectin (10μg/mL) to each well that will be used. Incubate for 10-15 minutes at room temperature. 2. Prepare siRNA/Lipofectamine RNAiMAX (Invitrogen) complexes for reverse transfection according to manufacturer s instructions. Use the recommended amounts for one well of a 24-well dish per chamber to be used (100μL total). An analogous product/protocol for siRNA transfection can be substituted. 3. Remove the fibronectin from the wells of the chambered coverglass. Add 100μL of siRNA/Lipofectamine RNAiMAX complexes to each well that will be used. 4. Aliquot 20,000 histone H2B-mCherry-expressing cells (in 500μL) per well containing transfection mixture. 5. Allow cells to incubate for ~24 hours before replacing transfection mixture with 1mL of regular cell culture medium. 6. Incubate cells an additional 24 hours before image acquisition (48 hours post-transfection). Microscope setup and image acquisition7. There are three main parts to the image acquisition setup: 1) a cell incubator that fits on the microscope stage and maintains a constant humid environment of 37°C, and 5% CO 2 ; 2) an inverted microscope equipped with an automated ...

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Imaging the division process in living tissue culture cells

Cited by 76 publications

References 61 publications

Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells

Cell Adhesion Molecule L1 Disrupts E-Cadherin-Containing Adherens Junctions and Increases Scattering and Motility of MCF7 Breast Carcinoma Cells

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Time-lapse Imaging of Mitosis After siRNA Transfection

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