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An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

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Immunoblot analysis with affinity-purified antiBSN UC56, 372, and 176 antibodies. (A) Total proteins  were isolated from mouse  testis and skin, and samples  were resolved by electrophoresis through 8.5% SDS– polyacrylamide gels. Proteins  were transferred by electroblotting to Immobilon-P  membranes (Millipore Corp.,  Bedford, MA), and blots were  subjected to immunoblot  analysis as described by the  manufacturer. Each of the three anti-BSN peptide antibodies were affinity column purified before use. In both testis and skin samples, a  single band of 120 kD was detected; this band was not detected by secondary antibody alone or by preimmune sera. (B) Total proteins  were isolated from a variety of adult mouse tissues and subjected to immunoblot analysis as outlined in A. Abbreviations are as indicated in the legend to Fig. 3, except: hEP, human epidermal keratinocytes; BR, brain.
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Figure 6: Immunoblot analysis with affinity-purified antiBSN UC56, 372, and 176 antibodies. (A) Total proteins were isolated from mouse testis and skin, and samples were resolved by electrophoresis through 8.5% SDS– polyacrylamide gels. Proteins were transferred by electroblotting to Immobilon-P membranes (Millipore Corp., Bedford, MA), and blots were subjected to immunoblot analysis as described by the manufacturer. Each of the three anti-BSN peptide antibodies were affinity column purified before use. In both testis and skin samples, a single band of 120 kD was detected; this band was not detected by secondary antibody alone or by preimmune sera. (B) Total proteins were isolated from a variety of adult mouse tissues and subjected to immunoblot analysis as outlined in A. Abbreviations are as indicated in the legend to Fig. 3, except: hEP, human epidermal keratinocytes; BR, brain.

Mentions: Both BSN1a and BSN1b cDNAs are predicted to encode 120-kD polypeptides; however, this has never been confirmed by immunoblot analysis. We therefore raised monospecific rabbit antibodies to three different peptide sequences present in the shared regions of these proteins (see Materials and Methods). As judged by immunoblot analysis, each of the three different affinity-purified peptide antibodies (UC56, 372, and 176) detected a single crossreacting band of 120 kD in protein extracts from mouse testis (Fig. 6 A). A band of this size was also detected in protein extracts from mouse skin and from other epithelial tissues known to contain keratinocytes (Fig. 6, A and B). Collectively, these findings: (a) establish the size of basonuclin in testis, skin, and other stratified epithelia; (b) verify the specificity of our antisera; and (c) suggest that, if other major forms of basonuclins exist, they either must be 120 kD in size or alternatively must have a most peculiar splice pattern, missing three different domains of the basonuclin protein.


An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Immunoblot analysis with affinity-purified antiBSN UC56, 372, and 176 antibodies. (A) Total proteins  were isolated from mouse  testis and skin, and samples  were resolved by electrophoresis through 8.5% SDS– polyacrylamide gels. Proteins  were transferred by electroblotting to Immobilon-P  membranes (Millipore Corp.,  Bedford, MA), and blots were  subjected to immunoblot  analysis as described by the  manufacturer. Each of the three anti-BSN peptide antibodies were affinity column purified before use. In both testis and skin samples, a  single band of 120 kD was detected; this band was not detected by secondary antibody alone or by preimmune sera. (B) Total proteins  were isolated from a variety of adult mouse tissues and subjected to immunoblot analysis as outlined in A. Abbreviations are as indicated in the legend to Fig. 3, except: hEP, human epidermal keratinocytes; BR, brain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139879&req=5

Figure 6: Immunoblot analysis with affinity-purified antiBSN UC56, 372, and 176 antibodies. (A) Total proteins were isolated from mouse testis and skin, and samples were resolved by electrophoresis through 8.5% SDS– polyacrylamide gels. Proteins were transferred by electroblotting to Immobilon-P membranes (Millipore Corp., Bedford, MA), and blots were subjected to immunoblot analysis as described by the manufacturer. Each of the three anti-BSN peptide antibodies were affinity column purified before use. In both testis and skin samples, a single band of 120 kD was detected; this band was not detected by secondary antibody alone or by preimmune sera. (B) Total proteins were isolated from a variety of adult mouse tissues and subjected to immunoblot analysis as outlined in A. Abbreviations are as indicated in the legend to Fig. 3, except: hEP, human epidermal keratinocytes; BR, brain.
Mentions: Both BSN1a and BSN1b cDNAs are predicted to encode 120-kD polypeptides; however, this has never been confirmed by immunoblot analysis. We therefore raised monospecific rabbit antibodies to three different peptide sequences present in the shared regions of these proteins (see Materials and Methods). As judged by immunoblot analysis, each of the three different affinity-purified peptide antibodies (UC56, 372, and 176) detected a single crossreacting band of 120 kD in protein extracts from mouse testis (Fig. 6 A). A band of this size was also detected in protein extracts from mouse skin and from other epithelial tissues known to contain keratinocytes (Fig. 6, A and B). Collectively, these findings: (a) establish the size of basonuclin in testis, skin, and other stratified epithelia; (b) verify the specificity of our antisera; and (c) suggest that, if other major forms of basonuclins exist, they either must be 120 kD in size or alternatively must have a most peculiar splice pattern, missing three different domains of the basonuclin protein.

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Show MeSH
Related in: MedlinePlus