Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium.

Generation of transgenic

Xenopus laevis expressing photoactivatable green fluorescent protein (PAGFP) A plasmid containing the coding sequence of PAGFP ( Patterson and Lippincott-Schwartz, 2002 ) was provided by G.H. Patterson and J. Lippincott-Schwartz (National Institutes of Health [NIH], Bethesda, MD). Transgenic Xenopus laevis were generated using the REMI method ( Kroll and Amaya, 1996 ). In brief, the coding sequence of PAGFP was placed downstream from the Xenopus opsin promoter, which confined expression specifically to the rod photoreceptors ( Mani et al., 2001 ). The plasmid was linearized with XhoI endonuclease and incubated with isolated Xenopus laevis sperm nuclei that were similarly digested. Eggs were fertilized by injection of the sperm nuclei. Transgenic embryos were identified by epifluorescent fundus imaging and allowed to develop into tadpoles and adult frogs.

Tissue preparation

Xenopus tadpoles, stage 42–60, expressing PAGFP in rods were dark-adapted for at least 2 h before experiment. All subsequent procedures were performed under infrared illumination to minimize activation of the light receptor, rhodopsin. Tadpoles were anesthetized by bathing in 0.05% tricaine (Ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich) and decapitated. Eyes were removed and retinas were dissected into frog Ringer’s solution (in mM: 120 NaCl, 2 KCl, 10 HEPES, 1.6 MgCl 2 , 10 glucose, 0.03 EDTA, and 1.0 CaCl 2 ). Retinas were oriented ganglion cell side down in a 50-µl bubble of Ringer’s on a polypropylene sheet and sliced into strips ∼50–100-µm wide and 100–200-µm long. Slices were transferred to an imaging chamber that consisted of a 35-mm polystyrene Petri dish, into the center of which a 5-mm diameter hole was drilled and then covered with a No. 1 glass coverslip attached with tackywax, which formed the bottom of the chamber. A moist sponge was placed in the Petri dish and the lid was loosely applied, such that humidity prevented evaporation of the Ringer’s solution while allowing free gas exchange. The chamber was then place onto the stage of the inverted confocal/multiphoton microscope. All procedures and experiments were performed at 21°C within 2 h of retinal dissection. Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Multiphoton photoconversion of PAGFP and imaging in live Xenopus rods Imaging and protein flux measurements were performed with a custom-built confocal/multiphoton microscope described previously ( Peet et al., 2004 ; Calvert et al., 2007 ) that was modified by the addition of a custom LabVIEW-based software interface that allows for rapid (up to 12 Hz) image acquisition and protocols that control the laser intensity and positioning for photoconversion of fluorescent molecules (designed and implemented in conjunction with Michael Coleman, Coleman Technologies Inc.). Transgenic expression of proteins under the Xenopus opsin promoter results in heterogeneous protein levels across rods in a given animal’s retina ( Peet et al., 2004 ). Thus, 3-D scans of the retinal slices were performed before experiment to identify rods with sufficient PAGFP expression and that were well oriented with their long axis as close to parallel to the x–y image plane as possible. The initial 3-D scans were performed with visible, confocal scanning using the 488-nm line of an argon-ion laser (model 163C; Newport Corp.) focused to the diffraction limit with a 60×, 1.2 NA, water-immersion objective (Plan Apo VC; Nikon). The 3-D coordinates for the photoconversion pulse were manually selected from these initial scans using the LabVIEW interface. PAGFP was photoconverted at the specified coordinates by multiphoton excitation from the Ti:S laser (Mai Tai HP; Newport Corp.) tuned to 820 nm and focused to the diffraction limit. The use of multiphoton excitation allowed the production of spatially well-defined fields of photoconverted molecules ( Zipfel et al., 2003 ) and minimized the exposure of the tissue to visible light. The laser exposures ranged from 0.1 to 100 ms and 10 to 20 mW (average power). The equilibration of the photoconverted PAGFP was then monitored with serial x–y scans with 488-nm confocal excitation that intersected the photoconversion site. Rapid focus corrections were made before and after the photoconversion pulse to account for the measured focus difference between 820- and 488-nm illumination caused by objective chromatic aberration ( Calvert et al., 2007 ). Raw images were processed using custom MATLAB (The MathWorks) routines to correct for slight field inhomogeneities inherent to the optical system and for nonlinearities in the photon detectors as follows. Full field scans with each laser in the system of solutions of fluorescein were averaged to generate image field–flattening maps. Except where noted, no other image processing was done. The 3-D point spread functions ( psf ) were estimated from spatial fluorescence distribution patterns obtained during 3-D scans of 0.1-µm fluorescent microspheres (Polysciences, Inc.), as described previously ( Calvert et al., 2007 ). The fluorescence profiles in x , y and x , z were approximated by peak normalized Gaussians with σ x,y = 0.16 and σ z 1 = σ z 2 = 0.68 for the focused Ti:sapphire laser tuned to 820 nm (multiphoton excitation, cf. Eq. 6 in Theory section), and σ x,y = 0.16 and σ z = 0.61 for the 488-nm line of the argon ion laser. Online supplemental material Although the approaches used to model the diffusional movement of PAGFP in the rod are presented in the Theory section, additional theoretical details of the methods used to obtain numerical solutions to equations, process and fit the data with theoretical curves generated by the model, as well as video clips of PAGFP diffusing in the IS and OS compartments and equilibrating throughout the rod cytoplasm are available at http://www.jgp.org/cgi/content/full/jgp.200910322/DC1 .

Online supplemental material Although the approaches used to model the diffusional movement of PAGFP in the rod are presented in the Theory section, additional theoretical details of the methods used to obtain numerical solutions to equations, process and fit the data with theoretical curves generated by the model, as well as video clips of PAGFP diffusing in the IS and OS compartments and equilibrating throughout the rod cytoplasm are available at http://www.jgp.org/cgi/content/full/jgp.200910322/DC1 .