Limits...
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.

Show MeSH

Related in: MedlinePlus

Subcellular localization of CALM and syntaxin2 as revealed by immunofluorescence staining. (A, C, D) SMAP2(+/+) and (−/−) germ cells were prepared as in Figure 3 and stained with anti-SMAP2, anti-CALM, anti-syntaxin2, anti-sp56, anti-TGN38, and DAPI, as indicated. The 1:1 mix is a mixed sample of SMAP2(+/+) and (−/−) cells. Pachytene spermatocytes and round spermatids are shown. Bar, 5 μm. (B) Immunoprecipitation of CALM. Lysates prepared from wild-type testes were immunoprecipitated with an anti-CALM antibody, and the precipitates were blotted with anti-CALM, anti-SMAP2, or anti-syntaxin2, as indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756916&req=5

Figure 7: Subcellular localization of CALM and syntaxin2 as revealed by immunofluorescence staining. (A, C, D) SMAP2(+/+) and (−/−) germ cells were prepared as in Figure 3 and stained with anti-SMAP2, anti-CALM, anti-syntaxin2, anti-sp56, anti-TGN38, and DAPI, as indicated. The 1:1 mix is a mixed sample of SMAP2(+/+) and (−/−) cells. Pachytene spermatocytes and round spermatids are shown. Bar, 5 μm. (B) Immunoprecipitation of CALM. Lysates prepared from wild-type testes were immunoprecipitated with an anti-CALM antibody, and the precipitates were blotted with anti-CALM, anti-SMAP2, or anti-syntaxin2, as indicated.

Mentions: The proacrosomal vesicles budding from the TGN in SMAP2(−/−) spermatids had larger diameters than those in the wild-type cells (Figure 5). CALM is a protein that affects the size of clathrin-coated vesicles, since vesicles become enlarged in cells lacking CALM/AP180 (Zhang et al., 1998; Nonet et al., 1999; Meyerholz et al., 2005). In addition, we previously reported a physical association between SMAP2 and CALM (Natsume et al., 2006). Therefore, to explore a possible link between SMAP2 and the vesicle size, we performed double immunofluorescence of SMAP2 and CALM using a germ cell suspension (Figure 7A). In wild-type spermatocytes and round spermatids, SMAP2 was concentrated at the TGN, as described earlier, whereas CALM fluorescence was detected diffusely in the cytoplasm but also colocalized with SMAP2. In the SMAP2(−/−) cells, CALM was no longer localized to the TGN (the overall amount of CALM was not affected by SMAP2 deficiency; data not shown). When the wild-type and SMAP2(−/−) germ cells were mixed at a 1:1 ratio, CALM was colocalized with SMAP2 at the TGN in the wild-type cells but homogenously distributed in the SMAP2(−/−) cells. Thus mobilization of CALM to the TGN appears to be dependent on SMAP2.


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)

Subcellular localization of CALM and syntaxin2 as revealed by immunofluorescence staining. (A, C, D) SMAP2(+/+) and (−/−) germ cells were prepared as in Figure 3 and stained with anti-SMAP2, anti-CALM, anti-syntaxin2, anti-sp56, anti-TGN38, and DAPI, as indicated. The 1:1 mix is a mixed sample of SMAP2(+/+) and (−/−) cells. Pachytene spermatocytes and round spermatids are shown. Bar, 5 μm. (B) Immunoprecipitation of CALM. Lysates prepared from wild-type testes were immunoprecipitated with an anti-CALM antibody, and the precipitates were blotted with anti-CALM, anti-SMAP2, or anti-syntaxin2, as indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756916&req=5

Figure 7: Subcellular localization of CALM and syntaxin2 as revealed by immunofluorescence staining. (A, C, D) SMAP2(+/+) and (−/−) germ cells were prepared as in Figure 3 and stained with anti-SMAP2, anti-CALM, anti-syntaxin2, anti-sp56, anti-TGN38, and DAPI, as indicated. The 1:1 mix is a mixed sample of SMAP2(+/+) and (−/−) cells. Pachytene spermatocytes and round spermatids are shown. Bar, 5 μm. (B) Immunoprecipitation of CALM. Lysates prepared from wild-type testes were immunoprecipitated with an anti-CALM antibody, and the precipitates were blotted with anti-CALM, anti-SMAP2, or anti-syntaxin2, as indicated.
Mentions: The proacrosomal vesicles budding from the TGN in SMAP2(−/−) spermatids had larger diameters than those in the wild-type cells (Figure 5). CALM is a protein that affects the size of clathrin-coated vesicles, since vesicles become enlarged in cells lacking CALM/AP180 (Zhang et al., 1998; Nonet et al., 1999; Meyerholz et al., 2005). In addition, we previously reported a physical association between SMAP2 and CALM (Natsume et al., 2006). Therefore, to explore a possible link between SMAP2 and the vesicle size, we performed double immunofluorescence of SMAP2 and CALM using a germ cell suspension (Figure 7A). In wild-type spermatocytes and round spermatids, SMAP2 was concentrated at the TGN, as described earlier, whereas CALM fluorescence was detected diffusely in the cytoplasm but also colocalized with SMAP2. In the SMAP2(−/−) cells, CALM was no longer localized to the TGN (the overall amount of CALM was not affected by SMAP2 deficiency; data not shown). When the wild-type and SMAP2(−/−) germ cells were mixed at a 1:1 ratio, CALM was colocalized with SMAP2 at the TGN in the wild-type cells but homogenously distributed in the SMAP2(−/−) cells. Thus mobilization of CALM to the TGN appears to be dependent on SMAP2.

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