A FRAP-Based Method for Monitoring Molecular Transport in Ciliary Photoreceptor Cells In Vivo
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The outer segment of rod and cone photoreceptor cells represents a highly modified primary sensory cilium. It renews on a daily basis throughout lifetime and effective vectorial transport to the cilium is essential for the maintenance of the photoreceptor cell function. Defects in molecules of transport modules lead to severe retinal ciliopathies. We have recently established a fluorescence recovery after photobleaching (FRAP)-based method to monitor molecular trafficking in living rodent photoreceptor cells. We irreversibly bleach the fluorescence of tagged molecules (e.g. eGFP-Rhodopsin) in photoreceptor cells of native vibratome sections through the retina by high laser intensity. In the laser scanning microscope, the recovery of the fluorescent signal is monitored over time and the kinetics of movements of molecules can be quantitatively ascertained.
Key wordsCiliary transport Primary sensory cilia Ciliated photoreceptor cells Rhodopsin Intracellular transport
The present study was supported by the German Research Council (DFG) FOR 2149/WO548-8, EC FP7/2009/241955 (SYSCILIA), the FAUN Foundation, Nurnberg, and the JGU Research Support (Stage 1). We thank Dr. J.H. Wilson, Houston, TX, for kindly providing the hRho-eGFP knock-in mouse line and Dr. Helen May-Simera for critical comments on the manuscript.
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