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D.W. Anderson, D.S. Papermaster; Expression of Moesin–GFP Fusion Proteins in Transgenic X. laevis Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1709.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Moesin possesses an N–terminal FERM domain that binds membranes and a C–terminal actin–binding domain. When it is activated, the protein can anchor vesicles to the cytoskeleton and may play a key role in the transport and fusion of rhodopsin transport carriers (RTCs) to the base of the connecting cilium. Previously the dominant negative rab8T22N mutant was shown to interfere with docking of RTCs to the outer segment (Moritz et al. MBC 12:2341,'01; Anderson et al. ARVO '04). Deretic et al. (MBC 15:359, '04) showed that moesin colocalizes with rab8 on RTCs and that propranalol abolishes this association resulting in the accumulation of undocked vesicles beneath the connecting cilium similar to the effect of rab8 T22N. We are investigating the impact of expression of GFP–tagged full–length moesin and its fragments upon vesicle trafficking in transgenic X. laevis. Methods:An X. laevis moesin clone (xMsn) was obtained by RT–PCR from retinal mRNA. A cDNA clone encoding the FERM domain of xMsn was created by PCR. The Xenopus opsin promotor drove expression of xMsn–GFP fusion proteins in transgenic X. laevis as described by Moritz et al.( IOVS 40:3276, '99). Tadpole eyes were frozen sectioned and visualized by confocal microscopy after counterstaining the glycoproteins with Texas–red conjugated to wheat germ agglutinin. Results:Full–length moesin–GFP fusion protein (xMsn 580–GFP) has a cytoplasmic distribution with an extension into the connecting cilium but is undetected in calycal processes. It is also present in the synapse. A construct with amino acids 1–320 fused to GFP (xMsn 320–GFP) is a moesin fragment with a FERM domain, but no actin binding domain. This protein prominently decorates calycal processes and inner segment plasma membrane. It is mostly above the nucleus with a faint signal at the synapse. High expression of the transgene appears to be associated with a loss of central rods as demonstrated by larger distances and more prevalent cones between rods when compared to control retinas. Apoptosis assays are underway to ascertain if the central rods are dying. EM studies are underway to determine if there is a block in vesicle docking. Conclusions:The loss of rods from the xMsn 320–GFP retinas is not as dramatic as that seen with rab8T22N indicating that photoreceptors may be partially able to compensate for the interference caused by xMsn 320–GFP but can not tolerate the effects of rab8T22N.
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