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G. H. Grossman, G. Hoppe, G. J. T. Pauer, S. A. Hagstrom; Compartment-Specific Tulp1 Interactions in Phototreceptors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4067.
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Mutations in TULP1 cause autosomal recessive retinitis pigmentosa in humans and retinal degeneration in mice. Tulp1 is photoreceptor-specific protein that is known to play a role in synaptic function in the outer plexiform layer (OPL), and in protein transport from the inner segment (IS) to the outer segment (OS). To study the multiple functions of Tulp1, our strategy is to identify and distinguish Tulp1 interacting proteins in these two photoreceptor compartments.
We employed time-controlled transcardiac perfusion cross-linking to stabilize Tulp1 complex partners in WT mice. Laser microdissection was then used to isolate and capture the IS and OPL compartments, as well as samples from the inner plexiform layer (IPL), an internal Tulp1-free control. Western blot analyses using an anti-Tulp1 antibody were performed on whole retinal lysate and compartment-specific samples (IS, OPL and IPL) to detect Tulp1-positive complexes. Results were compared to control retinas that did not receive crosslinking treatment.
A signal corresponding to Tulp1 (~70 kDa) was detected in the non-crosslinked and crosslinked retinal lysates, as well as crosslinked IS and OPL specific samples. Two additional bands at ~160 and 250 kDa were detected in the crosslinked whole retinal lysate. The 250 kDa band was also present in the IS but not in the OPL isolated crosslinked tissues. No high molecular weight Tulp1-positive complexes were detected in the non-crosslinked retinas.
Distinct bands in the higher molecular weight range observed in our crosslinked samples likely represent Tulp1 functional complexes. The differences in Tulp1-positive complexes between IS and OPL suggest that Tulp1 participates in multiple pathways in the photoreceptor cell via assembly into compartment-specific macromolecular structures. Ongoing proteomic analysis of these bands should clarify the different roles that Tulp1 plays in the IS and OPL.
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