Absence of SCAPER causes male infertility in humans and Drosophila by modulating microtubule dynamics during meiosis.

BACKGROUND: Mutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies. METHODS: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy. RESULTS: We show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs. CONCLUSIONS: Our results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.

SCAPER localizes to primary cilia and its mutation affects cilia length, causing Bardet-Biedl syndrome.

Studies of ciliopathies have served in elucidating much of our knowledge of structure and function of primary cilia. We report humans with Bardet-Biedl syndrome who display intellectual disability, retinitis pigmentosa, obesity, short stature and brachydactyly, stemming from a homozyogous truncation mutation in SCAPER, a gene previously associated with mitotic progression. Our findings, based on linkage analysis and exome sequencing studies of two remotely related large consanguineous families, are in line with recent reports of SCAPER variants associated with intellectual disability and retinitis pigmentosa. Using immuno-fluorescence and live cell imaging in NIH/3T3 fibroblasts and SH-SY5Y neuroblastoma cell lines over-expressing SCAPER, we demonstrate that both wild type and mutant SCAPER are expressed in primary cilia and co-localize with tubulin, forming bundles of microtubules. While wild type SCAPER was rarely localized along the ciliary axoneme and basal body, the aberrant protein remained sequestered to the cilia, mostly at the ciliary tip. Notably, longer cilia were demonstrated both in human affected fibroblasts compared to controls, as well as in NIH/3T3 cells transfected with mutant versus wildtype SCAPER. As SCAPER expression is known to peak at late G1 and S phase, overlapping the timing of ciliary resorption, our data suggest a possible role of SCAPER in ciliary dynamics and disassembly, also affecting microtubule-related mitotic progression. Thus, we outline a human ciliopathy syndrome and demonstrate that it is caused by a mutation in SCAPER, affecting primary cilia.