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The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis.

Funaki T, Kon S, Tanabe K, Natsume W, Sato S, Shimizu T, Yoshida N, Wong WF, Ogura A, Ogawa T, Inoue K, Ogonuki N, Miki H, Mochida K, Endoh K, Yomogida K, Fukumoto M, Horai R, Iwakura Y, Ito C, Toshimori K, Watanabe T, Satake M - Mol. Biol. Cell (2013)

Bottom Line: In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis.Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation.Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Immunology, Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.

ABSTRACT
The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.

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Expression and subcellular location of SMAP2 during spermatogenesis as detected by immunofluorescence staining. Single-cell suspensions were prepared from seminiferous tubules of wild-type testes, centrifuged onto glass slides, and fixed. (A) The cells were stained with anti-SMAP2, anti-γH2AX, and DAPI. Based on the staining pattern of the nuclear γH2AX and DAPI, differentiation stages were assigned as indicated. (B) The cells were stained as indicated. Pachytene spermatocytes and round spermatids are shown. Clathrin is the vesicle marker, TGN38 and syntaxin6 are TGN markers, and GM130 is a cis-Golgi marker. Bar, 5 μm. Values of r (average ±SD) represent the Pearson's coefficients quantifying the degree of colocalization between SMAP2 and the various marker proteins. For r > 0.5, colocalization is significant.
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Figure 3: Expression and subcellular location of SMAP2 during spermatogenesis as detected by immunofluorescence staining. Single-cell suspensions were prepared from seminiferous tubules of wild-type testes, centrifuged onto glass slides, and fixed. (A) The cells were stained with anti-SMAP2, anti-γH2AX, and DAPI. Based on the staining pattern of the nuclear γH2AX and DAPI, differentiation stages were assigned as indicated. (B) The cells were stained as indicated. Pachytene spermatocytes and round spermatids are shown. Clathrin is the vesicle marker, TGN38 and syntaxin6 are TGN markers, and GM130 is a cis-Golgi marker. Bar, 5 μm. Values of r (average ±SD) represent the Pearson's coefficients quantifying the degree of colocalization between SMAP2 and the various marker proteins. For r > 0.5, colocalization is significant.

Mentions: SMAP2 expression during spermatogenesis was examined by immunofluorescence using a testicular cell suspension that was spun down onto microscope slides (Figure 3A). 4′,6-Diamidino-2-phenylindole (DAPI) staining showed the nuclear morphology, as well as chromatin condensation, whereas γH2AX staining detected sites of DNA double-strand breaks. Combined use of DAPI and γH2AX allowed the identification of the stages of spermatogenesis (Xu, 2003; Figure 3A). SMAP2 expression was not detected in spermatogonia or in leptotene spermatocytes but could first be detected in the cytoplasm of zygotene spermatocytes as multiple punctae. In pachytene spermatocytes and round spermatids, SMAP2 fluorescence appeared not only as multiple punctae but also in a single, large focus. SMAP2 fluorescence was absent in elongated spermatids and sperm. Thus SMAP2 expression was stage dependent. Immunofluorescence localization of SMAP2 is consistent with the detection by Northern blot (Figure 1C).


The Arf GAP SMAP2 is necessary for organized vesicle budding from the trans-Golgi network and subsequent acrosome formation in spermiogenesis.

Funaki T, Kon S, Tanabe K, Natsume W, Sato S, Shimizu T, Yoshida N, Wong WF, Ogura A, Ogawa T, Inoue K, Ogonuki N, Miki H, Mochida K, Endoh K, Yomogida K, Fukumoto M, Horai R, Iwakura Y, Ito C, Toshimori K, Watanabe T, Satake M - Mol. Biol. Cell (2013)

Expression and subcellular location of SMAP2 during spermatogenesis as detected by immunofluorescence staining. Single-cell suspensions were prepared from seminiferous tubules of wild-type testes, centrifuged onto glass slides, and fixed. (A) The cells were stained with anti-SMAP2, anti-γH2AX, and DAPI. Based on the staining pattern of the nuclear γH2AX and DAPI, differentiation stages were assigned as indicated. (B) The cells were stained as indicated. Pachytene spermatocytes and round spermatids are shown. Clathrin is the vesicle marker, TGN38 and syntaxin6 are TGN markers, and GM130 is a cis-Golgi marker. Bar, 5 μm. Values of r (average ±SD) represent the Pearson's coefficients quantifying the degree of colocalization between SMAP2 and the various marker proteins. For r > 0.5, colocalization is significant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756916&req=5

Figure 3: Expression and subcellular location of SMAP2 during spermatogenesis as detected by immunofluorescence staining. Single-cell suspensions were prepared from seminiferous tubules of wild-type testes, centrifuged onto glass slides, and fixed. (A) The cells were stained with anti-SMAP2, anti-γH2AX, and DAPI. Based on the staining pattern of the nuclear γH2AX and DAPI, differentiation stages were assigned as indicated. (B) The cells were stained as indicated. Pachytene spermatocytes and round spermatids are shown. Clathrin is the vesicle marker, TGN38 and syntaxin6 are TGN markers, and GM130 is a cis-Golgi marker. Bar, 5 μm. Values of r (average ±SD) represent the Pearson's coefficients quantifying the degree of colocalization between SMAP2 and the various marker proteins. For r > 0.5, colocalization is significant.
Mentions: SMAP2 expression during spermatogenesis was examined by immunofluorescence using a testicular cell suspension that was spun down onto microscope slides (Figure 3A). 4′,6-Diamidino-2-phenylindole (DAPI) staining showed the nuclear morphology, as well as chromatin condensation, whereas γH2AX staining detected sites of DNA double-strand breaks. Combined use of DAPI and γH2AX allowed the identification of the stages of spermatogenesis (Xu, 2003; Figure 3A). SMAP2 expression was not detected in spermatogonia or in leptotene spermatocytes but could first be detected in the cytoplasm of zygotene spermatocytes as multiple punctae. In pachytene spermatocytes and round spermatids, SMAP2 fluorescence appeared not only as multiple punctae but also in a single, large focus. SMAP2 fluorescence was absent in elongated spermatids and sperm. Thus SMAP2 expression was stage dependent. Immunofluorescence localization of SMAP2 is consistent with the detection by Northern blot (Figure 1C).

Bottom Line: In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis.Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation.Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Immunology, Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.

ABSTRACT
The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.

Show MeSH
Related in: MedlinePlus