Multiphoton GFP photoconversion reveals cilia diffusion dynamics

Researchers measured protein diffusion through a sensory cilium. Courtesy of Peter D. Calvert.

Cilia are present on most cells and play important functions in vision, hearing, and smell. Scientist want to know more about the unknown functions of many cilia because mutations in genes that encode cilia proteins can cause serious diseases such as Bardet-Biedl syndrome, Usher syndrome, poly-cystic kidney disease, and retinal degeneration.

Cilia don’t synthesize proteins or membrane, and research is focused on how proteins are delivered to and removed from these organelles. Researchers believe that cilia impose selective barriers to the movement of proteins but haven’t been able to show this experimentally because some of the cilia’s structures have diameters near or below the resolution of light microscopy.

But now, a team of researchers led by Peter Calvert at SUNY Upstate Medical University along with researchers from Lehigh University and the University of California, Davis have measured the mobility of photoactivatable GFP in the connecting cilium of live Xenopus retinal rod photoreceptors and in subcellular compartments bridged by the connecting cilium. In addition, the team measured the overall time for the protein concentration to equilibrate within and between compartments.

The researchers photoconverted GFP at specified coordinates using multiphoton excitation from a Ti:sapphire laser tuned to 820 nm and focused to the diffraction limit. The multiphoton excitation produced spatially well-defined fields of photoconverted molecules and minimized the tissue’s exposure to visible light. They then used confocal excitation at 488 nm to monitor the equilibration of the photoconverted GFP with serial x–y scans that intersected the area of photoconversion.

The results showed that in the connecting cilium diffusion was somewhat slower than expected in an aqueous cell compartment, but that this structure was not a major barrier to protein diffusion. However, the diffusion in the inner and outer segments bridged by the connection cilium was found to be substantially slower than in other cells studied. The authors offer various explanations of what might cause this slower diffusion.

Research Paper:

The Journal of General Physiology, Vol. 135, No. 3, 173-196 Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium

Peter D. Calvert, William E. Schiesser, and Edward N. Pugh, Jr.