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D.W. Anderson, A.M. Provenzano, D. Deretic, D.S. Papermaster; A Moesin–GFP Fusion Protein Induces Degeneration of Rods in Transgenic Xenopus Laevis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3728.
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© ARVO (1962-2015); The Authors (2016-present)
Moesin (Msn) is a FERM domain protein of 580 amino acids that can bind membranes with its N–terminal domain and actin with its C–terminal domain, when activated by phosphorylation at T561. The activated protein can anchor vesicles to the cytoskeleton and may play a key role in the transport and fusion of vesicles to the base of the rod connecting cilium. Propranolol has been shown to disrupt the transport of rhodopsin bearing vesicles probably by reducing the PI4,5P2 content of vesicle bilayers so that Msn is no longer associated with them (Deretic et al., 2004). This results in an accumulation of undocked vesicles near the base of the connecting cilium. We obtained an X. laevis Msn cDNA clone with the goal of creating a dominant negative transgene to study the role of Msn in rhodopsin transport (Anderson et al. ARVO, 2005).
The X. laevis Msn protein, the T561D–mutant protein and N– and C–terminal domains were expressed as GFP fusion proteins under the control of a 1.3 kb xenopus opsin promotor as described by Moritz et al. (IOVS 40:3276, 1999). Fusion proteins using the N–terminal FERM domain had GFP attached to the C–termini. C–terminal Msn constructs had GFP attached to the N–termini. Transgenic tadpoles were fixed in 4% paraformaldehyde in the afternoon, and retinas were visualized by confocal microscopy of frozen sections.
The T561D mutation of Msn makes a constitutively active protein by mimicking phosphorylation at that site. Although the retinas of 14 dpf T561D–GFP tadpoles appear normal, GFP can be detected in the RPE indicating that apoptotic rods have been phagocytosed. At 21 dpf and especially by 30 dpf, degeneration of rods is widespread; the degree of damage appears to be dependent on the expression level of the transgene. The T561D–GFP fusion protein decorates the actin bundles extending from the outer limiting membrane into the calycal processes indicating that it associates with the actin cytoskeleton. Expression of the N–terminal FERM domain does not result in a degenerative phenotype even at 90 dpf. The C–terminal actin–binding domain is also tolerated in rods.
Expression of the Msn T561D–GFP fusion protein results in apoptosis of the rods. The mechanism underlying this phenotype is not yet apparent. At this time, there is no evidence of undocked vesicles caused by expression of that transgene.
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