Single-cell kinase activity measurements of the mouse retina by two-photon ex vivo imaging.

Abstract

Purpose : We previously established a number of genetically-coded fluorescent probes to visualize the activity of a specific kinase in living cells based on Förster resonance energy transfer (FRET biosensor) and generated mouse strains which express a specific FRET biosensor ubiquitously. This study examined visualization of kinase activities at single-cell resolution in the live retinal explant expressing the FRET biosensor.

Methods : Retinas expressing FRET biosensors designed for extracellular signal-regulated kinase (ERK), protein kinase A (PKA) and AMP-activated protein kinase (AMPK) were examined. The retinas were isolated, flat-mounted on a culture insert, and perfused with Ames' solution. When comparing rods and cones, the retinal explants were incubated with rhodamine labeled peanuts agglutinin (rhodamine-PNA) before the measurements to label cones specifically. The explants were then time-lapsed imaged with a multi-photon microscope. Z-stack images were acquired using a z-stack interval of 1 μm to observe each layer of the retina. Kinase activity were visualized based on FRET efficiency. To examine drug responses, the retina was perfused with drug solution (forskolin for PKA sensor and 2-deoxy-D-glucose for AMPK sensor).

Results : The retinal explants were imaged at cellular resolution in all layers of the retina; even small photoreceptor cells (about 1-2 μm in diameter) were imaged at single-cell resolution. Cones were distinguished from rods as rhodamine-PNA positive cells. Retinal cells were time-lapse imaged for hours without significant degeneration. Robust upregulation of kinase activities and subsequent recovery were observed when the retina was perfused with drug and then normal Ames' solution, respectively.

Conclusions : Kinese activities were visualized at single-cell resolution in the FRET biosensor expressing retina. This imaging method provides precise spatial infomation for the regulation of kinase activities in response to a stimulation (drug perfusion, light exposure, etc.). For example, rods and cones in the mouse retina can be compared directly to study cone-specific kinase regulation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Ex vivo live imaging of the mouse reitna expressing FRET biosensor designed for ERK. Images of each layer were acquired by changing the position of the objective lens and thus obtained z-stack images were used to reconstruct a longitudinal image of the retina.