Abstract
Abstract: :
Purpose: Rab8 is a small GTPase involved in transport of post–Golgi vesicles. Previous work with wild type (wt) canine rab8 (crab8), a dominant negative mutant, T22N, and a constitutively active mutant, Q67L, in transgenic Xenopus laevis rods demonstrated that rab8 is required for transport and docking of rhodopsin bearing vesicles to the outer segment (Moritz et al. MBC 12:2341,’01). The mutant proteins caused defective vesicle transport and induced apoptosis of the photoreceptors. To determine if species variations between the canine and Xenopus proteins could result in different observed phenotypes, the endogenous X. laevis rab8 (xrab8wt) was isolated and compared to crab8wt. Xrab8 and its associated mutants will be used to further define components of vesicle transport and study the process of apoptosis in photoreceptors. Methods: X. laevis rab8 clones were obtained by RT–PCR from retinal mRNA, and the mutations created by site–directed–mutagenesis. Transgenic X. laevis expressing wt and mutant xrab8 GFP fusion proteins under control of the Xenopus opsin promotor were generated as described by Moritz et al., IOVS 40:3276, ’99. Tadpoles were raised in cyclic light and sacrificed between 14 and 21 days post fertilization (dpf). Sections were visualized by confocal microscopy. Crab8 T22N and xrab8 T22N transgenic animals were also raised under constant light for 21 dpf to determine if light could protect the photoreceptors from apoptosis. Constant light exposure protects rods expressing the K44A mutation of the GTPase Dynamin I from apoptosis (Xu et al., ARVO 2001) since synaptic vesicles are not recycling in hyperpolarized rods. Results: There are 2 rab8 genes expressed in X. laevis with 17 nucleotide differences between them but no amino acid changes. There are 10 amino acid changes between xrab8 and crab8, of which one is non–conservative. Wild type xrab8 displays the same intracellular distribution as wt crab8. The xrab8 T22N and the Q67L mutants also result in the same phenotype as their respective crab8 constructs. The xrab8 T22N mutation results in an early degeneration of the rods, depleting the central retina completely by 14 dpf in most cases. Exposure to constant light did not prevent retinal degeneration in either the xrab8 T22N or crab8 T22N animals. The xrab8 Q67L mutation results in a later degeneration of the rods, with some surviving to 21 dpf. Conclusions: Xrab8, crab8 and their mutants produce identical results in transgenic X. laevis, with no differences that could be observed in this system. The mechanism causing the rab8 T22N mutant rods to undergo apoptosis is not light dependent.
Keywords: photoreceptors • transgenics/knock–outs • apoptosis/cell death