RC/BTB2 is a binding partner of sperm associated antigen 16S (SPAG16S), which is regulator of spermiogenesis in mice, a process during which sperm flagella are formed. The expression of Rc/btb2 is also regulated by multicilin, a protein that controls ciliogenesis. Given that mouse Rc/btb2 mRNA is not only expressed in tissues bearing motile cilia, but also in tissues without motile cilia, we investigated whether RC/BTB2 plays a role in the general process of ciliogenesis by studying two somatic cells lines that have primary cilia, NIH3T3 and IMCD3. We discovered that the subcellular localization of RT/BTB2 in the NIH3T3 and IMCD3 cells encompasses the pathway for ciliogenesis. RC/BTB2 was found in the Golgi bodies and centrosomes, two key structures essential for normal ciliogenesis. Knockdown of Rc/btb2 gene expression in these cell lines disrupted ciliogenesis. The percentage of cells with primary cilia was significantly reduced in stable cell lines transduced with specific Rc/btb2 shRNA viruses compared to the control cells. When cilia were formed in the knockdown cells, they were significantly shorter than those in the control cells. Knockdown of Rc/btb2 expression did not affect cell proliferation and the cell cycle. Exogenous expression of RC/BTB2 in these stable knockdown cells restored ciliogenesis. These findings suggest that RC/BTB2 is a necessary component of the process of formation of primary cilia in somatic cells, perhaps through the transportation of cargos from Golgi bodies to centrosomes for cilia assembling.
The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell-type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non-motile cilia, and other microtubule-associated cellular functions.
cilia; flagella; axoneme; central pair complex
Sperm-associated antigen 6 (Spag6) gene, which encodes an axonemal protein (SPAG6), ubiquitously expresses in tissue and organs containing ciliated cells. The present work was to investigate whether SPAG6 expressed in cochlear hair cells and, if so, to explore the presumable correlations between prestin and SPAG6. The distribution of SPAG6 in organ of Corti and the morphological features of hair cells in basilar membrane were investigated by immunofluorescent staining. The amount of prestin in Spag6 mutant mice was measured by Western blotting and real-time PCR, respectively. Additionally, co-immunoprecipitation tests were performed to confirm the presumed interaction between prestin and SPAG6. We observed that SPAG6 expressed in the cuticular plate in outer hair cells (OHCs) and prestin in the lateral wall of OHCs that located along with SPAG6 at this site. In comparison to Spag6 +/+ mice, Spag6 −/− mice showed apparent morphological abnormity of OHCs and lower intensity of prestin fluorescence. The expression of prestin in Spag6 −/− mice reduced significantly at both protein and mRNA levels. Moreover, co-immunoprecipitation tests demonstrated the interaction between prestin and SPAG6. Taken together, these data indicate that SPAG6 is indispensible for the stability of OHCs by maintaining the normal expression of prestin, which implies that Spag6 gene is essential for mechanosensory function of OHCs.
Sperm associated antigen 6; Spag6; Prestin; Outer hair cells; OHCs; Gene knockout mice
The product of the meiosis-expressed gene 1 (MEIG1) is found in the cell bodies of spermatocytes and recruited to the manchette, a structure unique to elongating spermatids, by Parkin co-regulated gene (PACRG). This complex is essential for targeting cargo to the manchette during sperm flagellum assembly. Here we show that MEIG1 adopts a unique fold that provides a large surface for interacting with other proteins. We mutated 12 exposed and conserved amino acids and show that four of these mutations (W50A, K57E, F66A, Y68A) dramatically reduce binding to PACRG. These four amino acids form a contiguous hydrophobic patch on one end of the protein. Furthermore, each of these four mutations diminishes the ability of MEIG1 to stabilize PACRG when expressed in bacteria. Together these studies establish the unique structure and key interaction surface of MEIG1 and provide a framework to explore how MEIG1 recruits proteins to build the sperm tail.
Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were observed in the Spag6-deficient MEF cultures. The percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs developed multiple cilia. Furthermore, SPAG6 selectively increased expression of acetylated tubulin, a microtubule stability marker. The Spag6-deficient MEFs were more sensitive to paclitaxel, a microtubule stabilizer. Our studies reveal new roles for SPAG6 in modulation of cell morphology, proliferation, migration, and ciliogenesis.
Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.
A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.
Summary: In the manchette, a structure at the base of the elongating spermatid, the proteins MEIG1 and PACRG act in a complex to control cargo transport and direct formation of the flagellum.
Cargo transport; Manchette; Sperm flagella; Spermiogenesis
The C–H activation of methane, ethane, and t-butane on inner and outer surfaces of nitrogen-doped carbon nanotube (NCNTs) are investigated using density functional theory. It includes NCNTs with different diameters, different N and O concentrations, and different types (armchair and zigzag). A universal structure-reactivity relationship is proposed to characterize the C–H activation occurring both on the inner and outer surfaces of the nano channel. The C–O bond distance, spin density and charge carried by active oxygen are found to be highly related to the C–H activation barriers. Based on these theoretical results, some useful strategies are suggested to guide the rational design of more effective catalysts by nano channel confinement.
Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.
SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.
Currently very little is known about the differential expression and function of the transcription factor SOX5 during B cell maturation. We identified two new splice variants of SOX5 in human B cells, encoding the known L-SOX5B isoform and a new shorter isoform L-SOX5F. The SOX5 transcripts are highly expressed during late stages of B-cell differentiation, including atypical memory B cells, activated CD21low B cells and germinal center B cells of tonsils. In tonsillar sections SOX5 expression was predominantly polarized to centrocytes within the light zone. After in vitro stimulation, SOX5 expression was down-regulated during proliferation while high expression levels were permissible for plasmablast differentiation. Overexpression of L-SOX5F in human primary B lymphocytes resulted in reduced proliferation, less survival of CD138neg B cells, but comparable numbers of CD138+CD38hi plasmablasts compared to control cells. Thus, our findings describe for the first time a functional role of SOX5 during late B cell development reducing the proliferative capacity and thus potentially affecting the differentiation of B cells during the germinal center response.
Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen–17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with “9 + 2” motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.
primary ciliary dyskinesia; axoneme; central pair; Spag17; cilia
SPAG16 is a critical structural component of motile cilia and flagella. In the eukaryotic unicellular algae Chlamydomonas, loss of gene function causes flagellar paralysis and prevents assembly of the “9 + 2” axoneme central pair. In mice, we have previously shown that loss of Spag16 gene function causes male infertility and severe sperm motility defects. We have also reported that a heterozygous mutation of the human SPAG16 gene reduces stability of the sperm axonemal central apparatus.
In the present study, we analyzed DNA samples from 60 infertile male volunteers of Western European (Italian) origin, to search for novel SPAG16 gene mutations, and to determine whether increased prevalence of SPAG16 single nucleotide polymorphisms (SNPs) was associated with infertility phenotypes. Semen parameters were evaluated by light microscopy and sperm morphology was comprehensively analyzed by transmission electron microscopy (TEM).
For gene analysis, sequences were generated covering exons encoding the conserved WD40 repeat region of the SPAG16 protein and the flanking splice junctions. No novel mutations were found, and the four SNPs in the assessed gene region were present at expected frequencies. The minor alleles were not associated with any assessed sperm parameter in the sample population.
Analysis of the SPAG16 regions encoding the conserved WD repeats revealed no evidence for association of mutations or genetic variation with sperm motility and ultrastructural sperm characteristics in a cohort of Italian infertile males.
Sperm ultrastructure; Axoneme; Motile cilia; Male infertility; Central apparatus; Semen analysis
Mouse RC/BTB2 is an unstudied protein of the RCC1 (Regulator of Chromosome Condensation) superfamily. Because of the significant remodeling of chromatin that occurs during spermiogenesis, we characterized the expression and localization of mouse RC/BTB2 in the testis and male germ cells. The Rc/btb2 gene yields two major transcripts: 2.3 kb Rc/btb2-s, present in most somatic tissues examined; and 2.5 kb Rc/btb2-t, which contains a unique non-translated exon in its 5′-UTR that is only detected in the testis. During the first wave of spermatogenesis, Rc/btb2-t mRNA is expressed from day 8 after birth, reaching highest levels of expression at day 30 after birth. The full-length protein contains three RCC1 domains in the N-terminus, and a BTB domain in the C-terminus. In the testis, the protein is detectable from day 12, but is progressively up-regulated to day 30 and day 42 after birth. In spermatids, some of the protein co-localizes with acrosomal markers sp56 and peanut lectin, indicating that it is an acrosomal protein. A GFP-tagged RCC1 domain is present throughout the cytoplasm of transfected CHO cells. However, both GFP-tagged, full-length RC/BTB2 and a GFP-tagged BTB domain localize to vesicles in close proximity to the nuclear membrane, suggesting that the BTB domain might play a role in mediating full-length RC/BTB2 localization. Since RCC1 domains associate with Ran, a small GTPase that regulates molecular trafficking, it is possible that RC/BTB2 plays a role in transporting proteins during acrosome formation.
A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. Single chondrocytes and chondrons isolated from bovine articular cartilage were compressed by micromanipulation between two parallel surfaces in order to investigate their biomechanical properties and to discover the mechanical significance of the PCM. The force imposed on the cells was measured directly during compression to various deformations and then holding. When the nominal strain at the end of compression was 50 per cent, force relaxation showed that the cells were viscoelastic, but this viscoelasticity was generally insignificant when the nominal strain was 30 per cent or lower. The viscoelastic behaviour might be due to the mechanical response of the cell cytoskeleton and/or nucleus at higher deformations. A finite-element analysis was applied to simulate the experimental force-displacement/time data and to obtain mechanical property parameters of the chondrocytes and chondrons. Because of the large strains in the cells, a nonlinear elastic model was used for simulations of compression to 30 per cent nominal strain and a nonlinear viscoelastic model for 50 per cent. The elastic model yielded a Young's modulus of 14 ± 1 kPa (mean ± s.e.) for chondrocytes and 19 ± 2 kPa for chondrons, respectively. The viscoelastic model generated an instantaneous elastic modulus of 21 ± 3 and 27 ± 4 kPa, a long-term modulus of 9.3 ± 0.8 and 12 ± 1 kPa and an apparent viscosity of 2.8 ± 0.5 and 3.4 ± 0.6 kPa s for chondrocytes and chondrons, respectively. It was concluded that chondrons were generally stiffer and showed less viscoelastic behaviour than chondrocytes, and that the PCM significantly influenced the mechanical properties of the cells.
chondrocyte; chondron; finite-element modelling; micromanipulation; nonlinear elasticity; viscoelasticity
Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.
The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action. In addition, the passive flagella, inhibited by sodium metavanadate to disable the dyneins, were examined for mechanical abnormalities. Live Spag16L-null sperm exhibited much less bending of the flagellum during the beat. The amount of microtubule sliding in the R-bend direction of the beat was selectively restricted, which suggests that there is limited activation of the dyneins on one side of the axoneme in the live cells. This is corroborated by the results on detergent-extracted sperm models. The flagellar response to calcium is greatly reduced. The calcium response requires the activation of the dyneins on outer doublets 1, 2, 3, and 4. These are the same dyneins required for R-bend formation. In axonemes prepared to disintegrate by microtubule sliding, we observed little or no extrusion of doublets 1 and 2, consistent with a reduced activity of their dyneins. This deficit in motor function, and an increased rigidity of the midpiece region which we detected in the passive flagella, together can explain the observed motility characteristics of the Spag16L-null sperm.
Mouse sperm lacking SPAG16L have altered motility and a reduced response to calcium.
axoneme; calcium; central pair; ciliopathies; dynein; hyperactivation
Overexpression of sphingosine kinase-1 (SPHK1) has been demonstrated to be associated with the development and progression in various types of human cancers. The current study was to characterize the expression of SPHK1 in salivary gland carcinomas (SGC) and to investigate the association between SPHK1 expression and progression of SGC.
The expression of SPHK1 was examined in 2 normal salivary gland tissues, 8 SGC tissues of various clinical stages, and 5 pairs of primary SGC and adjacent salivary gland tissues from the same patient, using real-time PCR and western blot analysis. Furthermore, the SPHK1 protein expression was analyzed in 159 clinicopathologically characterized SGC cases by immunohistochemistry. Statistical analyses were performed to determine the prognostic and diagnostic associations.
SPHK1 expression was found to be markedly upregulated in SGC tissues than that in the normal salivary gland tissues and paired adjacent salivary gland tissues, at both mRNA and protein levels. Statistical analysis revealed a significant correlation of SPHK1 expression with the clinical stage (P = 0.005), T classification (P = 0.017), N classification (P = 0.009), M classification (P = 0.002), and pathological differentiation (P = 0.013). Patients with higher SPHK1 expression had shorter overall survival time, whereas patients with lower SPHK1 expression had better survival. Importantly, patients in the group without adjuvant therapy who exhibited high SPHK1 expression had significantly lower overall survival rates compared with those with low SPHK1 expression. Moreover, multivariate analysis suggested that SPHK1 expression might be an independent prognostic indicator for the survival of SGC patients.
Our results suggest that SPHK1 expression is associated with SGC progression, and might represent as a novel and valuable predictor for adjuvant therapy to SGC patients.
SOX5 is a transcription factor with homology to the high mobility group box region of the testis-determining factor, SRY. Both the mouse and human SOX5 genes encode a 48-kDa SOX5 protein (S-SOX5) that is only present in tissues containing cells with motile cilia/flagella. The mammalian sperm-associated antigen 6 gene (SPAG6) encodes an axoneme central apparatus protein. Because human and mouse SPAG6 gene promoters contain multiple potential binding sites for SOX5, SPAG6 gene regulation by S-SOX5 was investigated in BEAS-2B cells, a line derived from human bronchial cells. Like FOXJ1, a transcription factor known to be essential for motile ciliogenesis, S-SOX5 stimulated mouse and human SPAG6 promoter function in BEAS-2B cells, but the effect was abrogated when the SOX5 binding sites were mutated or deleted. S-SOX5 and FOXJ1 functioned cooperatively in stimulating SPAG6 promoter activity. The SPAG6 message was up-regulated when S-SOX5 was overexpressed in BEAS-2B cells, and silencing of S-SOX5 by RNA interference down-regulated SPAG6 transcripts. Chromatin immunoprecipitation and EMSA experiments demonstrated that S-SOX5 associates with the SPAG6 promoter directly. The present study demonstrates that SPAG6 is a S-SOX5 target gene, indicating a key role for S-SOX5 in the formation and function of motile cilia.
DNA-Protein Interaction; Gene Regulation; Promoters; Site-directed Mutagenesis; Sperm; Central Apparatus; Motile Cilia; SOX5; SPAG6; Transcription Regulation
Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.
SPAG16L; kinase; phosphorylation; sperm motility
Targeted deletion of Tssk1 & 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser285 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS′ N-terminus in enzyme interaction, identify Ser 285 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 & 2 in spermiogenesis.
Haploinsufficiency; infertility; gene targeting deletion; TSSK1; TSSK2; TSKS; spermatozoa; protein interaction; protein phosphorylation
Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 & 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of TSSK 1 & 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.
TSSK2; TSKS; Centriole; Spermatid; Spermatozoa; Spermiogenesis; Flagellogenesis
cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.