Deciphering the structure and function of Als2cr4 in the mouse retina

Abstract

Purpose: The role of Als2cr4 (amyotrophic lateral sclerosis 2 [juvenile] chromosome region, candidate 4; also known as hypothetical protein FLJ33282) in the mouse retina was determined by characterizing the molecular structure, cellular interacting partners, and potential biochemical functions. Previous in situ hybridization and gene expression profiles show that the mRNAs encoding Als2cr4 are abundant in the eye, hippocampus, cerebellum, and olfactory bulb.

Methods: From predicted antigenic epitopes of Als2cr4, two novel antibodies were developed to examine protein expression and morphologic localization in retinas from light-adapted and dark-adapted mice by immunohistochemistry, immunoblot analysis, and immunoelectron microscopy, and then immunoprecipitation was performed to identify interacting proteins by mass spectroscopy.

Results: Peptide antibodies with Als2cr4 antigenic epitopes from either the amino- or carboxyl terminus were characterized with Als2cr4 recombinant proteins and peptide competition assays. Als2cr4 is a 45-kDa insoluble protein, highly enriched in retina, and localizes to photoreceptor outer segments, ciliary complex, and horizontal cells in the outer plexiform layer. Immunoelectron microscopy for Als2cr4 verified its expression in the discs of photoreceptor outer segments. Immunoprecipitation and mass spectroscopy identified eight potential interacting partners: vimentin, actin, myosin Va, myosin VI, myosin X, myosin XIV, kinesin 1, Als2cr4, and lamin B-1.

Conclusions: Als2cr4 is a novel protein, with a probable tetraspanin-like membrane structure, that is localized in photoreceptors and in the postsynaptic outer plexiform layer and that interacts with cytoskeletal proteins. Als2cr4 may be involved in membrane transport between the photoreceptor inner and outer segments and may be a key component in maintaining the structural integrity of the outer segment.

Figure 1.

Computer-modeled 2-D structure. An Als2cr4-translated AA sequence was used to identify transmembrane helices with version 2 of the TMHMM Server. The data were input into TMRPres2D software to create a 2-D model of α-helical or β-barrel transmembrane proteins. Four potential transmembrane domains were identified, in addition to a long amino-terminal tail (226 AAs) and a short carboxyl-terminal tail (30 AAs), which are predicted to reside within the cytoplasm.

Figure 2.

IB analysis of Als2cr4 protein expression. Each lane contains 20 μg of total retinal lysate from P30 mice. Control WT, Nrl^−/−, Arr4^−/−, Arr1^−/− or Arr-DKO mice were light or dark adapted and killed, and retinal lysates were prepared. The proteins were resolved on 10% SDS-PAGE, transferred onto PVDF membrane, probed with the pAb FLJ-LG followed by the secondary antibody, goat anti-rabbit HRP-conjugated IgG, and processed for ECL. As a loading control, the membrane was probed with the mAb GAPDH and the appropriate secondary antibody.

Figure 3.

Als2cr4 immunohistochemical localization. P30 WT mice were either (A) light or (B) dark adapted and killed. The retina sections (7 μm) were incubated with pAb FLJ-FM followed by incubation with Alexa Fluor-488 (Als2cr4) goat anti-rabbit IgG. The nuclei were stained with TO-PRO3 iodide. Top: magnified region of the photoreceptor layer. Middle: view of the retina at ×63 magnification. Bottom: magnified region of the OPL. Results demonstrate a similar staining pattern for Als2cr4 regardless of lighting condition. Punctate staining was observed in the photoreceptor layer, whereas staining in the OPL was more uniform. Magnification, ×63.

Figure 4.

Dual IHC of Als2cr4 and Arr1 in retinas from light- or dark-adapted mice. P30 WT mice were either (A) light- or (B) dark-adapted and killed. The retina sections were dual stained with mAb D9F2 for Arr1 and pAb FLJ-FM for Als2cr4. Secondary antibodies were Alexa Fluor-568 goat anti-mouse IgG (Arr1) and Alexa Fluor-488 goat anti-rabbit IgG (Als2cr4). Nuclei were stained with TO-PRO3 iodide. (A, top) A magnified region of the merged photoreceptor layer in the bottom panel demonstrates that Als2cr4 was present in the connecting cilia. After 1 hour of light exposure, Arr1 localization was exclusive to the OS, whereas Als2cr4 was present in both the OS and connecting cilia. (B) Al2cr4 was localized in the postsynaptic OPL, with no co-localization of Arr1 with Als2cr4 in rod spherules or cone pedicles. Magnification, ×63.

Figure 5.

Dual IHC localization of Als2cr4 and calbindin in horizontal cells. P30 WT mice were either (A) light- or (B) dark-adapted and killed. The retinal sections were dual stained with mAb calbindin and pAb FLJ-FM. Secondary antibodies were Alexa Fluor-568 goat anti-mouse IgG (Calbindin) and Alexa Fluor-488 goat anti-rabbit IgG (Als2cr4). The nuclei were stained with TO-PRO3 iodide. Top: magnified region of the photoreceptor layer. Middle: view of the retina at ×63 magnification. Bottom: magnified region of the OPL. White arrows denote dual immunohistochemical localization of Als2cr4 with calbindin at the OPL. Magnification, ×63.

Figure 6.

Immuno-EM analysis of Als2cr4 in WT retinas. Retinas from either (A) light- or (B) dark-adapted mice were identified with pAb FLJ-FM and secondary goat anti-rabbit-10 nm colloidal gold antibody particles. (A, B) Rod OS. IM-EM shows Als2cr4 embedded within the discs of photoreceptor OS. Magnification, ×20,000.

Figure 7.

Membrane localization of Als2cr4 transfected into HEK293 cells. Confocal microscopy images of 6xHis tagged-Als2cr4, transiently transfected into HEK293 cells. Als2cr4 subcellular localization was identified by immunohistochemical labeling with antibodies recognizing Als2cr4 with pAb FLJ-FM (green; a, b) or the His-tag with mAb 6xHis (red; c, d). Nucleic acids in the nuclei were labeled with TO-PRO 3 iodide. Cells transfected with a recombinant Als2cr4 cDNA construct expressed the recombinant Als2cr4 protein, which is localized to the cytoplasm and the plasma membrane, (f) whereas those transfected with no recombinant cDNA inserted into the pCDNA4/HisMax vector (e) exhibited no Als2cr4 expression, and the His-tag was limited to the nucleus. Labeling patterns of Als2cr4 pAb (b) and 6xHis mAb (d) displayed co-localization in the plasma membrane (f), thereby confirming Al2cr4 antibody specificity.

Figure 8.

IP with Als2cr4 and IB analysis verified interacting partners. The retinas of LA WT mice were used to immunoprecipitate novel Als2cr4-interacting partners. Individual membranes were probed with the indicated antibody (left) followed by secondary goat anti-rabbit HRP-conjugated antibody for ECL detection. Lane 1: experimental precleared beads; lane 2: experimental precleared supernatants; and lane 3: eluted antigens immunoprecipitated with 10 μg of FLJ-FM pAb. Results showed that Als2cr4 specifically immunoprecipitated myosin Va, myosin VI, and Arr4. The positive control showed that Als2cr4 was present in the eluted fraction (lane 3), and the negative control showed that Arr1 bound nonspecifically to the beads, was present in the supernatants, and did not interact with Als2cr4. Molecular mass markers (kilodaltons) are identified on the right. Bottom: light chain IgG.