The mitotic spindle protein SPAG5/Astrin connects to the Usher protein network postmitotically

Ferry Fj Kersten, Erwin van Wijk, Lisette Hetterschijt, Katharina Bauβ, Theo A Peters, Mariam G Aslanyan, Bert van der Zwaag, Uwe Wolfrum, Jan Ee Keunen, Ronald Roepman, Hannie Kremer, Ferry Fj Kersten, Erwin van Wijk, Lisette Hetterschijt, Katharina Bauβ, Theo A Peters, Mariam G Aslanyan, Bert van der Zwaag, Uwe Wolfrum, Jan Ee Keunen, Ronald Roepman, Hannie Kremer

Abstract

Background: Mutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina.

Results: We identified the centrosomal and microtubule-associated protein sperm-associated antigen (SPAG)5 in the retina. SPAG5 was also found to interact with another previously described USH2AisoB interaction partner: the centrosomal ninein-like protein NINLisoB. Using In situ hybridization, we found that Spag5 was widely expressed during murine embryonic development, with prominent signals in the eye, cochlea, brain, kidney and liver. SPAG5 expression in adult human tissues was detected by quantitative PCR, which identified expression in the retina, brain, intestine, kidney and testis. In the retina, Spag5, Ush2aisoB and NinlisoB were present at several subcellular structures of photoreceptor cells, and colocalized at the basal bodies.

Conclusions: Based on these results and on the suggested roles for USH proteins in vesicle transport and providing structural support to both the inner ear and the retina, we hypothesize that SPAG5, USH2AisoB and NINLisoB may function together in microtubule-based cytoplasmic trafficking of proteins that are essential for cilium formation, maintenance and/or function.

Figures

Figure 1
Figure 1
Interactions between sperm-associated antigen (SPAG)5 and Usher syndrome 2A isoform B (USH2AisoB). (A) Schematic protein structures of SPAG5 and the peptides encoded by deletion constructs. Numbers represent amino acids (aa; NP_006452). Protein fragments encoded by deletion constructs of SPAG5 and the intracellular domain (ICD) of USH2AisoB were used in a yeast two-hybrid assay, which identified a specific interaction between USH2AisoB (aa 5064 to 5196) and the C-terminal two coiled-coil domains (aa 973to 1193) of SPAG5. (B) Glutathione S-transferase (GST) pull-down assays showing that Flag-tagged SPAG5 is efficiently pulled down by GST-USH2AisoBICD, but not by GST alone, as detected by an anti-Flag antibody. The first lane shows 5% of the input of COS-1 cell lysate. (C) Hemagluttinin (HA)-SPAG5 aa 973-1193 from COS-1 lysates co-immunoprecipitated with GFP-fused USH2AisoBICD, but not with an unrelated HA-tagged protein (EPS8). As a positive control, HA-whirlin co-immunoprecipitated with green fluorescent protein (GFP)-fused USH2AisoBICD. The upper protein band in the middle blot represents the heavy chain of the anti-GFP antibody.
Figure 2
Figure 2
Validation of the interaction between sperm-associated antigen (SPAG)5 and ninein-like protein isoform B (NINLisoB) interaction. (A) The schematic protein structure of NINLisoB and the predicted intermediate filament domain (IF; aa 656-925), encoded by a deletion construct. A liquid β-galactosidase assay identified a specific interaction between SPAG5 (aa 973-1193) and the intermediate filament (IF) domain of NINLisoB. (B) Glutathione S-transferase (GST) pull-down assay showing that Flag-tagged NINLisoB was efficiently pulled down by GST-SPAG5 (aa 973-1193), but not by GST alone, as detected by an anti-Flag antibody. The first lane shows 5% of the protein input. (C) Flag-NINLisoB co-immunoprecipitated with full-length HA-SPAG5 and the positive control HA-USH2AisoB ICD, but not with the HA-tagged unrelated protein MDA5.
Figure 3
Figure 3
Expression of the gene sperm-associated antigen (SPAG)5 in human adult tissues and Spag5 expression during murine development. (A) Using quantitative PCR, SPAG5 expression was detected in the human retina, brain, duodenum, kidney and testis. (B-M). RNA in situ hybridization of Spag5 mRNA in mouse embryos at embryonic day (E)12.5-E18.5 and mouse eyes at postnatal day (P)7. (B-D) Spag5 was widely expressed during murine development (E12.5 to E16.5), with the most intense signals in the following structures (numbers and arrowheads) ventricular zone of forebrain (1), midbrain (2), liver (3), lung (4), kidney (5), adrenal gland (6), thymus gland (7), submandibular salivary gland (8), limbs (9) and eye (10). (E) Expression was also seen in the cochlea, primordial vibrissal follicles, upper and lower jaw, intestine, heart, olfactory epithelium and testis (data not shown). (D) From E16.5 onwards, expression was mainly detected in the eye, brain, kidney, lung, thymus, primordial vibrissal follicles and in the liver (data not shown). (F-K) A strong signal for Spag5 was seen in the eye during embryonic development E12.5-E18.5 in the neuroblastic layer of the retina. At (I) E16.5 and (K) E18.5, Spag5 expression was present in the developing inner nuclear layer (INL; 11) and, (L-M) although much weaker, in the ganglion cell layer (GCL; 12), and this was maintained at postnatal day 7.
Figure 4
Figure 4
Subcellular localization of sperm-associated antigen (Spag)5 in retina cryosections of adult (postnatal day (P)20) rats. (A-C) Co-immunostaining using(A) anti-SPAG5 (green) and (B) anti-pan-centrin (red, a marker for the connecting cilium, basal body and centrioles) antibodies. (C) Colocalization (C; yellow) was detected at the (Ci) basal body (BB) and (Cii) in one of the two centrioles of the inner nuclear layer (INL). (A,C) Centriole staining is also visible in the ganglion cell layer (GCL). In addition, weaker Spag5 labeling was detected at the outer limiting membrane (OLM; filled arrowhead), the outer plexiform layer (OPL), at the distal part of the connecting cilium (CC) and at the accessory centriole. (A-Ci-ii).High-magnification fluorescence microscopy of the BB/CC region and the INL.
Figure 5
Figure 5
Localization of sperm-associated antigen (Spag)5 by immunoelectron microscopy. Electron micrographs of pre-embedded anti-SPAG5 labeling in (A,B) longitudinal and (C) cross-sections of mouse rod photoreceptor cells. Spag5 was detected (A-B) around the basal body (BB), (A-C) in the collar-like extension (CE) of the apical inner segment; and (A) in some photoreceptors at the region of the rootlet (R). CC = connecting cilium; OS = outer segment; IS = inner segment. Scale bars: 0.5 μm.
Figure 6
Figure 6
Colocalization of sperm-associated antigen (Spag)5 and isoform B of Ush2a (Ush2aisoB) and ninein-like (NINLisoB) proteins in the retina of young adult (P20) rat. Co-immunostaining of Spag5 and Ush2aisoB in retinal cryosections using (A) anti-SPAG5 antibodies (red) and (B) anti-USH2AisoB antibodies (green) show (C) partial colocalization (yellow) at the basal body. Co-immunostaining using (D) anti-SPAG5 antibodies (green) and (E) anti-NINLisoB antibodies (red). (F) A partial colocalization (yellow) was detected at the basal body. In addition, both antibodies labeled (Di,Ei) the ciliary rootlets, which was confirmed by (I) co-staining (yellow) using (G) anti-NINLisoB (green) and (H) anti-rootletin (red) antibodies.
Figure 7
Figure 7
Specificity of anti-sperm-associated antigen (SPAG)5 antibodies. (A) Co-immunostaining of SPAG5 (green) and Usher syndrome 2A (USH2A; red) in retinal cryosections of adult (P20) rat using anti-USH2A and anti-SPAG5 antibodies. Both antibodies were blocked together with the SPAG5 antigen and after pre-absorption no specific SPAG5 signals were detected in contrast to the normal USH2A staining. (A') Western blot analysis of HA-tagged SPAG5 using (pre-absorbed) anti-hemagglutinin (HA) and anti-SPAG5 antibodies. After pre-absorption of the antibodies with the SPAG5 antigen, HA-SPAG5 could only be detected by anti-HA antibodies and not by the blocked anti-SPAG5 antibodies, indicating the specificity of the antibodies.

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