Analysis of Ligand-Receptor Interaction on the Surface of Living Cells by Fluorescence Correlation Spectroscopy~!2009-12-01~!2010-02-22~!2010-04-23~!

Miyagi, Hiraku; Maruyama, Ichiro

doi:10.2174/1874383801004010028

Cited by 8 publications

(7 citation statements)

References 14 publications

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“…The diffusion coefficient for EGF has been measured previously, both in 0.3% agarose 18 and in Hanks’ balanced salt solution, 19 and is roughly 1 × 10 −6 cm 2 /s in both cases; therefore, the agarose does not appreciably hinder the diffusion of EGF. The diffusion coefficient of CXCL12 in water is 1.74 × 10 −6 cm 2 /s.…”

Section: Methodssupporting

confidence: 51%

Improving the design of the agarose spot assay for eukaryotic cell chemotaxis

2014

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Migration of cells along gradients of effector molecules, i.e., chemotaxis, is necessary in immune response and is involved in development and cancer metastasis. The experimental assessment of chemotaxis thus is of high interest. The agarose spot assay is a simple tissue culture system used to analyze chemotaxis. Although direction sensing requires gradients to be sufficiently steep, how the chemical gradients developed in this assay change over time, and thus, under what conditions chemotaxis is plausible, has not yet been determined. Here, we use numerical solution of the diffusion equation to determine the chemoattractant gradient produced in the assay. Our analysis shows that, for the usual spot size, the lifetime of the assay is optimized if the chemoattractant concentration in the spot is initially 30 times the dissociation constant of the chemoattractant-receptor bond. This result holds regardless of the properties of the chemoattractant. With this initial concentration, the chemoattractant gradient falls to the minimum threshold for directional sensing at the same time that the concentration drops to the optimal level for detecting gradient direction. If a higher initial chemoattractant concentration is used, the useful lifetime of the assay is likely to be shortened because receptor saturation may decrease the cells’ sensitivity to the gradient; lower initial concentrations would result in too little chemoattractant for the cells to detect. Moreover, chemoattractants with higher diffusion coefficients would sustain gradients for less time. Based on previous measurements of the diffusion coefficients of the chemoattractants EGF and CXCL12, we estimate that the assay will produce gradients that cells can sense for a duration of 10 h for EGF and 5 h for CXCL12. These gradient durations are comparable to what can be achieved with the Boyden chamber assay. The analysis presented in this work facilitates determination of suitable parameters for the assay, and can be used to assess whether observed cell motility is likely due to chemotaxis or chemokinesis.

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Section: Methodssupporting

confidence: 51%

Improving the design of the agarose spot assay for eukaryotic cell chemotaxis

2014

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“…A DNA fragment encoding TrkB was cut out with Eco RI and Xba I enzymes from pTrkB-VN, and was then inserted into pBiFC-NLuc and pBiFC-CLuc, resulting in pTrkB-NLuc and pTrkB-CLuc, respectively. The construction of pGFP-TrkB was based on pEGFR-eGFP [ 30 ], and a DNA sequence encoding enhanced GFP, which was amplified by PCR using the primers 5'-CCGagatctATGGTGAGCAAGGGCGAGGAGCTGTTCACC (EGFP-F-BglII) and 5'-CCGgaattcCTTGTACAGCTCGTCCATGCCGAGAGTGATCC (EGFP-B-EcoRI), and was then inserted between Bgl II and Eco RI of pAEMXT-ACPwt-GPI (Covalys Bio-sciences, Witterswil, Switzerland), resulting in pAEMXT/GFP. A DNA sequence encoding human TrkB without its signal peptide (aa 1-31), but with (G 4 S) 3 , was inserted between Eco RI and Xho I of pAEMXT/GFP, resulting in pGFP-TrkB.…”

Section: Methodsmentioning

confidence: 99%

Brain-derived neurotrophic factor receptor TrkB exists as a preformed dimer in living cells

Shen

Maruyama

2012

JMS

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BackgroundNeurotrophins (NTs) and their receptors play crucial roles in the development, functions and maintenance of nervous systems. It is widely believed that NT-induced dimerization of the receptors initiates the transmembrane signaling. However, it is still controversial whether the receptor molecule has a monomeric or dimeric structure on the cell surface before its ligand binding.FindingsUsing chemical cross-linking, bimolecular fluorescence complementation (BiFC) and luciferase fragment complementation (LFC) assays, in this study, we show the brain-derived neurotrophic factor (BDNF) receptor TrkB exists as a homodimer before ligand binding. We have also found by using BiFC and LFC that the dimer forms in the endoplasmic reticulum (ER), and that the receptor lacking its intracellular domain cannot form the dimeric structure.ConclusionsMost, if not all, of the TrkB receptor has a preformed, yet inactive, homodimeric structure before BDNF binding. The intracellular domain of TrkB plays a crucial role in the spontaneous dimerization of the newly synthesized receptors, which occurs in ER. These findings provide new insight into an understanding of a molecular mechanism underlying transmembrane signaling mediated by NT receptors.

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“…In all cases we assumed a diffusion coefficient for EGF of 1×10 −6 cm 2 /s (Miyagi and Maruyama, 2010). We used a diffusion coefficient for fMLP of 4×10 −6 cm 2 /s, having estimated this value from the molecular weight of fMLP (Lauffenburger and Zigmond, 1981).…”

Section: Methodsmentioning

confidence: 99%

Determining whether observed eukaryotic cell migration indicates chemotactic responsiveness or random chemokinetic motion

Szatmary

Nossal

2017

Journal of Theoretical Biology

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Chemotaxis, the motion of cells directed by a gradient of chemoattractant molecules, guides cells in immune response, development, wound healing, and cancer. Unfortunately, this process is difficult to distinguish from chemokinesis, i.e., stimulated random cell motion. Chemotaxis is frequently inferred by determining how many cells cross a boundary in a chemotaxis assay, for example how many cells crawl into a chemoattractant-infused filter, or how many cells enter a defined region in an under-agarose assay or agarose spot assay. To mitigate possible ambiguity in whether motion observed in these assays is directed by the chemoattractant gradient or by chemokinesis, we developed a mathematical model to determine when such methods indeed indicate directed motion of cells. In contrast to previous analyses of chemotaxis assays, we report not just the gradients that arise in the assays but also resulting cell motion. We applied the model to data obtained from rigorous measurements and show, as examples, that MDA-MB-231 breast-cancer cells are at least 20 times less sensitive to gradients of EGF or CXCL12 than neutrophils are to formyl peptides; we then used this information to determine the extent to which gradient sensing increases the rate of boundary crossing relative to a random-motility control. Results show, for example, that in the filter assay, 2–4 times as many neutrophils pass through the filter when exposed to a gradient as when the gradient is absent. However, in the other combinations of cells and assays we considered, only 10–20% more cells are counted as having migrated in a directed, rather than random, motility condition. We also discuss the design of appropriate controls for these assays, which is difficult for the under-agarose and agarose spot assays. Moreover, although straightforward to perform with the filter assay, reliable controls are often not done. Consequently, we infer that chemotaxis is frequently over-reported, especially for cells like MDA-MB-231 cells, which move slowly and are relatively insensitive to gradients. Such results provide insights into the use of chemotaxis assays, particularly if one wants to acquire and analyze quantitative data.

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Analysis of Ligand-Receptor Interaction on the Surface of Living Cells by Fluorescence Correlation Spectroscopy~!2009-12-01~!2010-02-22~!2010-04-23~!

Cited by 8 publications

References 14 publications

Improving the design of the agarose spot assay for eukaryotic cell chemotaxis

Improving the design of the agarose spot assay for eukaryotic cell chemotaxis

Brain-derived neurotrophic factor receptor TrkB exists as a preformed dimer in living cells

Determining whether observed eukaryotic cell migration indicates chemotactic responsiveness or random chemokinetic motion

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