Monitoring cellular movementin vivowith photoconvertible fluorescence protein “Kaede” transgenic mice

Abstract

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4 ؉ T, CD8 ؉ T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.cell migration ͉ dendritic cells ͉ lymphocyte ͉ photoconvertible protein

Resident CD4+ T cells accumulate in lymphoid organs after prolonged antigen exposure

“…We established a photoconversion-based cell labelling method using the photoconvertible protein Dendra2, which emits green fluorescence in its native form (D-Green), but upon illumination with UV/violet light changes into a form that emits red fluorescence (D-Red). Photoconversion of cytosolic proteins has been reported to label cells for short time periods only due to fast protein turnover rates 14 . Therefore, we used a histone (H2B)-fused version of Dendra2 that allowed us to label cells for longer time periods by increasing protein stability.…”

“…Therefore, detection of resident cells in lymphoid organs required dedicated experimental approaches. Photoconvertible and photoactivatable proteins considerably advanced our tool box to study cell migration and have been used previously for cell tracking in LNs and PPs [12][13][14] . Yet, these reports analysed cell migration for hours or few days only.…”

“…Recently, the use of photoconvertible and photoactivatable proteins provided new insights into lymphocyte trafficking [12][13][14] . These fluorescent proteins change their emission wavelength upon exposure to light of specific wavelengths.…”

Generation of colonic IgA-secreting cells in the caecal patch

“…ALY/NscJcl-aly (aly/aly) and ICR mice were from CLEA Japan. Kaede-transgenic mice 29 were kindly provided by Dr Michio Tomura (Kyoto University). These mice were kept under specific-pathogen-free conditions at the Experimental Animal Facility, Graduate School of Medicine, Osaka University.…”

“…We analysed the mechanisms for the slower increase of colonic IgAsecreting cells in appendectomized mice using Kaede-transgenic mice, in which in vivo cell migration can be monitored 29 . Kaede is a photoconvertible protein, changing fluorescence from green to red with exposure of violet light.…”