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Lindsey Ebke, Gregory Grossman, Craig Beight, Gayle Pauer, Stephanie Hagstrom; Interaction of Tubby-like protein-1 (Tulp1) and Microtubule-Associated Proteins (MAPs) in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4197.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in microtubule-associated proteins (MAPs) have been shown to underlie neurodegenerative diseases, including retinitis pigmentosa (RP). MAP1A and MAP1B are expressed primarily in neurons where they function in stabilizing microtubules. Mutations in Tubby-like protein 1 (Tulp1) also cause RP. Tulp1 is a photoreceptor-specific protein that exhibits a genetic interaction with Mtap1a, whereby an allele of Mtap1a significantly attenuates photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins and Tulp1 have been implicated in protein transport through interactions with cytoskeletal elements. To investigate whether Tulp1 and MAP1 proteins associate in a functional complex in photoreceptor cells, we performed localization and interaction experiments.
Wild-type (wt) and tulp1-/- retinal tissue was examined by Western blot analysis, immunoprecipitation (IP), immunohistochemistry (IHC), and proximity ligand assay (PLA) using specific antibodies against MAP1A, MAP1B and Tulp1.
In wt photoreceptors, MAP1A is localized to the outer segments (OS) and MAP1B is present throughout all cellular compartments. In tulp1-/- retinas, MAP1A is absent from the OS and is mislocalized throughout the entire photoreceptor, while MAP1B is absent from the OS. Western blot analysis comparing wt and tulp1-/- retinal lysate shows that MAP1A and MAP1B protein levels are unaffected in the absence of Tulp1. IPs using wt retinal lysate and MAP1B antibodies shows that Tulp1 co-precipitates with MAP1B. In retinal sections, PLA analysis indicates that the Tulp1 / MAP1B interaction is localized throughout the photoreceptor inner segment and outer nuclear layer regions.
Tulp1 was identified as a MAP1B interacting protein by IP and PLA. In the tulp1-/- retina, MAP1A and MAP1B are no longer localized to the OS suggesting that MAP1 protein transport is Tulp1-dependent. Since MAP1A and MAP1B stabilize microtubules, their absence in the OS of tulp1-/- mice may destabilize microtubule networks in the photoreceptor, and thus be the underlying cause for the disorganized OSs and ultimately photoreceptor cell death in these mice.
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