Diffusion of a soluble protein,photoactivatable GFP,through a
sensory cilium |
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Authors: | Peter D Calvert William E Schiesser Edward N Pugh Jr |
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Institution: | 1.Department of Ophthalmology, and 2.Department of Biochemistry and Molecular Biology,
SUNY Upstate Medical University, Syracuse, NY 13210;3.Department of Chemical Engineering, Lehigh
University, Bethlehem, PA 18015;4.Department of Physiology, University of
California, Davis, Davis, CA 95616 |
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Abstract: | Transport of proteins to and from cilia is crucial for normal cell function and
survival, and interruption of transport has been implicated in degenerative and
neoplastic diseases. It has been hypothesized that the ciliary axoneme and
structures adjacent to and including the basal bodies of cilia impose selective
barriers to the movement of proteins into and out of the cilium. To examine this
hypothesis, using confocal and multiphoton microscopy we determined the mobility
of the highly soluble photoactivatable green fluorescent protein (PAGFP) in the
connecting cilium (CC) of live Xenopus retinal rod
photoreceptors, and in the contiguous subcellular compartments bridged by the
CC, the inner segment (IS) and the outer segment (OS). The estimated axial
diffusion coefficients are DCC = 2.8 ±
0.3, DIS = 5.2 ± 0.6, and
DOS = 0.079 ± 0.009
µm2 s−1. The results establish that the
CC does not pose a major barrier to protein diffusion within the rod cell.
However, the results also reveal that axial diffusion in each of the
rod’s compartments is substantially retarded relative to aqueous
solution: the axial diffusion of PAGFP was retarded ∼18-, 32- and
1,000-fold in the IS, CC, and OS, respectively, with ∼20-fold of the
reduction in the OS attributable to tortuosity imposed by the lamellar disc
membranes. Previous investigation of PAGFP diffusion in passed, spherical
Chinese hamster ovary cells yielded DCHO = 20
µm2 s−1, and estimating cytoplasmic
viscosity as Daq/DCHO
= 4.5, the residual 3- to 10-fold reduction in PAGFP diffusion is
ascribed to sub-optical resolution structures in the IS, CC, and OS
compartments. |
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Keywords: | |
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