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Distribution of sperm antigen 6 (SPAG6) and 16 (SPAG16) in mouse ciliated and non-ciliated tissues

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Abstract

The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.

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Funding

Funding for this work was provided by Fogarty International Center ‘National Institutes of Health’ Grant RO1TW006223. We thank Dr. Zhibing Zhang for kindly provide SPAG16 antibodies and María del Pilar Irigoyen (Facultad de Medicina, Universidad de la República) for her technical assistance.

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  1. Jimena Alciaturi and Gabriel Anesetti have equally contributed to this paper.

Authors and Affiliations

  1. Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, Montevideo, Uruguay

    Jimena Alciaturi, Gabriel Anesetti, Florencia Irigoin, Fernanda Skowronek & Rossana Sapiro

  2. Laboratorio de Genética Molecular Humana, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo, Uruguay

    Florencia Irigoin

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  1. Jimena Alciaturi
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Correspondence to Rossana Sapiro.

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10735_2019_9817_MOESM1_ESM.tif

Maps of Spag6/fluorescent proteins constructions. a) pEGFP-C2 Vector (Clontech Laboratories, Inc. A Takara Bio Company, CA USA, GenBank Accession: U576069) fused to the N-terminus of SPAG6 b) pDsRed-Express-N1 (Clontech Laboratories, Inc. A Takara Bio Company, CA USA) fused to the C-terminus of SPAG6. The figure was created using the free version of SnapGene® software. CMV: human cytomegalovirus immediate early enhancers, MCS: multiple cloning site, DsRed express: DsRed fluorescent protein,, EGFP: enhanced green fluorescent protein. Other vector data: The arrow indicates direction of (+) strand synthesis, SV40 enhancer and early promoter, SV40 origin of replication, SV40 polyadenylation signal, f1 bacteriophage origin of replication. Resistance to neomycin, kanamycin, and G418 (Geneticin) (TIF 8103 KB)

10735_2019_9817_MOESM2_ESM.tif

SPAG6 co-localizes with a subset of microtubule in CHO cells overexpressing SPAG6 and α-tubulin. Cells were co-transfected with pDsRed-N1/Spag6 and pAcGFP1-Tubulin and observed under an epifluorescence microscope. a SPAG6-RFP b Tubulin-GFP c Merged image Bars: 10 μm. (TIF 5994 KB)

10735_2019_9817_MOESM3_ESM.avi

Video showing sequential images of hTERT-RPE transfected cells show in Fig 7. Immunolocalization of acetylated and γ-tubulin. (AVI 36864 KB)

10735_2019_9817_MOESM4_ESM.tif

Confocal Z-stack projection of SPAG6-transfected NIH/3T3 cells and immunolocalization of γ-tubulin a Representative Z-stack projection of a group of cells where two transfected cells show EGFP- SPAG6 localization pattern. b Immunolabeling of γ-tubulin show the basal bodies and centrioles. Arrows indicate immunolabeling in transfected cells. c The merged image of both channels (red and green). No colocalization was visualized in any of the optical sections analyzed. Bars: 20 μm (TIF 3937 KB)

10735_2019_9817_MOESM5_ESM.tif

Endogenous SPAG6 immunolocalization in NIH/3T3 cultured cells. A very low signal of SPAG6 (green) is detected in the cytoplasm. No colocalization with γ-tubulin (red) or acetylated-tubulin (red) is observed. Yellow insets in bottom corner show a higher magnification of the sector indicated by the yellow square. An immunofluorescence image of cells without anti-SPAG6 antibody (negative control) is shown at the top left corner. Bars: 5 μm (TIF 3648 KB)

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Alciaturi, J., Anesetti, G., Irigoin, F. et al. Distribution of sperm antigen 6 (SPAG6) and 16 (SPAG16) in mouse ciliated and non-ciliated tissues. J Mol Hist 50, 189–202 (2019). https://doi.org/10.1007/s10735-019-09817-z

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  • DOI: https://doi.org/10.1007/s10735-019-09817-z

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