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Chemotaxis, the directed migration of cells towards the source of a soluble chemoattractant, plays a fundamental role in biological processes such as development and metastasis. Although there are numerous assays for chemotaxis—including the popular Boyden chamber, which measures cell migration through a permeable membrane—none is simultaneously inexpensive, easy to perform, suitable for altering conditions during the assay, and amenable to high-throughput multiplexing. In this issue, H. Wiggins and J. Rappoport from the University of Birmingham (Birmingham, UK) describe a new chemotaxis assay that possesses all of these advantages. The authors' “agarose spot” assay is a novel configuration of previous chemotaxis assays that use agarose as a medium in which a chemoattractant is suspended and can diffuse. They pipetted multiple small spots of a low–melting point agarose solution mixed with a chemoattractant onto a coverslip in the bottom of a tissue culture dish. The use of low–melting point agarose allows the addition of protein chemoattractants at a temperature that prevents their denaturation. Cells are then added to the tissue culture dish containing the coverslips with the agarose spots, allowed to adhere, and incubated overnight prior to microscopic analysis. Using epidermal growth factor (EGF) as the chemoattractant for an adherent breast cancer cell line, cell migration underneath the agarose spot was clearly observed using a light microscope, and verified by scanning electron microscopy images showing cells were underneath and not on top of agarose drops. Time-lapse imaging demonstrated direct and progressive movement of cells into the agarose spots, which is consistent with chemotaxis and not chemokinesis. Agarose spots with EGF had significantly more motile cells than control spots lacking EGF on the same coverslip, and the authors easily demonstrated that this chemotactic response was specific for EGF signaling by inhibiting it through the simple addition of the EGF receptor tyrosine kinase inhibitor AG1478 to the cell culture media. To show the high-throughput feasibility of their method, they examined five EGF-agarose spots per coverslip in each well of a six-well plate and found that the number of motile cells in the agarose spots was highly reproducible.
See “An agarose spot assay for chemotactic invasion”
MµiLive imaging of tissues and cells in culture has become an integral part of characterizing dynamic cellular processes. These experiments require maintenance of oxygen, nutrients, and temperature at physiological levels during imaging in order to be successful. Improvements in microscopy and cell culture techniques have led to the development of several culture devices, yet many of these instruments were designed to meet specific experimental needs. C.H. Picard, A. Donald, and colleagues at Cavendish Laboratory (Cambridge, UK) developed a modular microincubator (mµi) that facilitates a variety of imaging studies on tissue and cell culture samples. Their apparatus combines multiple features that have previously only been available in separate instruments, and is composed of modules that can be optimized to the particular needs of the user. The authors provide a detailed description of the construction of the reusable mµi, which allows for easy assembly and cleaning of the incubator. The incubator permits monolayer as well as 3-D culturing of cells, which can be seeded directly into the mµi or transferred from existing cultures. A cell monolayer may be cultured for 3 days and a tissue sample for 12 hours without circulation, allowing experiments to be conducted with constant culture conditions in the absence of the shear stresses present when using culture chips. Cells can be grown on any type of circular substrate of 500 µm or less in depth and imaged on upright or inverted microscopes since a gas-permeable membrane separates the culture medium from the environment. Temperature is controlled through the use of a Peltier module, which enables researchers to induce rapid temperature changes if desired or stabilize the temperature during the course of an experiment—such as when cells are exposed to oil from immersion objectives or when addition or changes of media are necessary. Media may be perfused through the mµi via capillary tubing and separated on either side of a thick tissue sample. The supplementary materials that accompany the article contain demonstrations of the capacity of the mµi in a broad spectrum of experiments including cell stretching, cell motility, and microrheology in variable environments.
See “A micro-incubator for cell and tissue imaging”
It's Not Easy Being GreenFluorescence microscopy is widely used as a non-destructive, highly sensitive technique for live cell and tissue imaging. Fluorescent dyes are available for detecting and localizing biomolecules, organelles, cell compartments, or specific tissues, while individual proteins can be studied using fluorescent reporters. Green plant tissues present unique challenges for fluorescence imaging applications due to the high levels of photosynthetic pigments that can block light penetration. In addition, plant tissues autofluoresce at the wavelengths commonly used in fluorescence microscopy and chloroplast vacuoles accumulate fluorescent metabolites that interfere with signal detection. To circumvent these problems, researchers led by I. Galis at the Max Planck Institute for Chemical Ecology demonstrated that silencing chlorophyll and xanthophyll pigments can enable improved fluorescence imaging in green plant tissues. The authors transiently silenced phytoene desaturase (PDS) in the leaves, stems, and floral tissues by virus-induced gene silencing (VIGS), leading to the photooxidative destruction of chlorophyll, and effectively bleaching green plant tissues. This change facilitated visualization of the sweet potato sporamin protein fused to enhanced GFP (eGFP) in the leaves of tobacco plants. PDS silencing reduced autofluorescence and led to more intense GFP signals in both the tobacco leaves and stems when compared with empty vector-treated controls. The authors found that PDS silencing altered the metabolic status of plants by reducing their photosynthetic capabilities. PDS-silenced plants were smaller than control plants. But the bleached leaves and stems were able to recruit sufficient nutrients from leaves that remained green and plants with only 3–4 mature green leaves were viable and able to produce flowers and seeds, although these seeds were unable to germinate and were produced in smaller quantities than in control plants. Even though the physiological status of the PDS-silenced plants was altered, the authors demonstrated that this did not impact the localization of fluorescently tagged proteins. VIGS silencing protocols have been established for several plant species, making this procedure potentially applicable to other types of plants as well.
Giving the Green Light to Cell Fusion
While fusion between different cell types has proved useful for generating hybridomas to produce monoclonal antibodies and for reprogramming somatic cell nuclei after fusion with embryonic stem cells, its in vivo role in various physiological and pathological conditions is currently of increasing interest. Cells fused in vitro can be visualized or isolated by introducing a marker into one cell type, while a different marker is used in another cell type. Fused cells can then be selected or observed by simultaneously expressing both markers. However, a limitation of this technique is that only fused cells that are tetraploid heterokaryons will be visualized or selected for, whereas fused cells that have lost one of the parental nuclei—or have resulted from the fusion of only cytoplasm from one of the cell types—will not. K. Pfannkuche and colleagues at the University of Cologne (Cologne, Germany) have devised a method for the visualization and selection of fused cells that overcomes this constraint. The authors stably transfected one cell type with a RFP/GFP double fluorescence indicator vector that constitutively expresses only RFP. Upon fusion with another cell type that has been stably transfected with a Cre recombinase expression vector, the Cre protein mediates a recombination event that deletes the loxP-flanked RFP gene from the indicator construct and activates expression of a bicistronic mRNA encoding the puromycin resistance gene and GFP. This allows visualization of fused cells that have switched from RFP to GFP expression and selection by puromycin. In instances where the Cre-expressing nucleus is lost, or when there is fusion of an indicator cell only with the cytoplasm from a Cre-expressing cell, it is still possible to visualize these fused cells containing only the nucleus with the indicator vector since they will also express GFP and the puromycin resistance gene. Cre activity can be controlled through the fusion of Cre with the estrogen receptor (ER). Treatment of cells containing the Cre-ER protein with 4-hydroxy-tamoxifen induces its nuclear translocation, which then induces Cre-mediated deletion of the RFP gene from the indicator construct.
