[Show abstract][Hide abstract] 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 was detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting revealed that SMAP2-deficient male mice were healthy and survived to adulthood, but were infertile and exhibited globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increased, TGN structures were distorted, acrosome formation was severely impaired, and reorganization of the nucleus did not proceed properly. CALM functions to regulate vesicle sizes, and this study showed that CALM was not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the SNARE complex, was not properly concentrated at the site of acrosome formation. Thus, the present 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.
Molecular biology of the cell 07/2013; 24(17). DOI:10.1091/mbc.E13-05-0234 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SMAP1 and SMAP2 proteins constitute a subfamily of the Arf-specific GTPase-activating proteins. Both SMAP proteins bind to clathrin heavy chains and are involved in the trafficking of clathrin-coated vesicles. In cells, SMAP1 regulates Arf6-dependent endocytosis of transferrin receptors from the coated pits of the plasma membrane, whereas SMAP2 regulates Arf1-dependent retrograde transport of TGN38 from the early endosome to the trans-Golgi network. The common and distinct features of SMAP1 and SMAP2 activity provide a valuable opportunity to examine the differential regulation of membrane trafficking by these two proteins. In this chapter, we describe several basic experimental procedures that have been used to study the regulation of membrane trafficking using SMAP proteins, including a GAP assay as well as procedures to study the transport of transferrin receptors and TGN38. In addition, a yeast two-hybrid system is described because of its utility in identifying novel molecules that interact with SMAP.
Methods in Enzymology 02/2008; 438:155-70. DOI:10.1016/S0076-6879(07)38011-7 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras-related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP-bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E-cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E-cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E-cadherin endocytosis should be one of the key elements in tumor cell biology.
Cancer Science 10/2006; 97(9):801-6. DOI:10.1111/j.1349-7006.2006.00251.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.
Molecular Biology of the Cell 07/2006; 17(6):2592-603. DOI:10.1091/mbc.E05-10-0909 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.
Molecular Biology of the Cell 05/2005; 16(4):1617-28. DOI:10.1091/mbc.E04-08-0683 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.
Journal of Experimental Medicine 08/2003; 198(1):51-61. DOI:10.1084/jem.20021200 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper discusses the impact of the hierarchical master-worker paradigm on performance of an application program, which solves an optimization problem by a parallel branch and bound algorithm on a distributed computing system. The application program, which this paper addresses, solves the BMI Eigenvalue Problem, which is an optimization problem to minimize the greatest eigenvalue of a bilinear matrix function. This paper proposes a parallel branch and bound algorithm to solve the BMI Eigenvalue Problem with the hierarchical master-worker paradigm. The experimental results showed that the conventional algorithm with the master-worker paradigm significantly degraded performance on a Grid test bed, where computing resources were distributed on WAN via a firewall; however, the hierarchical master-worker paradigm sustained good performance.
Cluster Computing and the Grid, 2003. Proceedings. CCGrid 2003. 3rd IEEE/ACM International Symposium on; 06/2003
[Show abstract][Hide abstract] ABSTRACT: Interleukin-10 (IL-10) is a T helper type 2 (Th2) cytokine that suppresses Th1-mediated, cell-mediated immune responses and reciprocally enhances antibody-mediated responses. Previous studies, however, demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. We then examined whether tumor-derived IL-10 could modulate systemic immune responses. Murine colon carcinoma (Colon 26) cells that were retrovirally transduced with the murine IL-10 gene (Colon 26/IL-10) were inoculated in syngeneic immunocompetent or T cell-defective nude mice. Growth of Colon 26/IL-10 tumors was augmented in immunocompetent and, to less extent, in nude mice compared with that of wild-type tumors developed in respective mice. Growth of wild-type tumors was accelerated to the same level as that of Colon 26/IL-10 tumors when wild type and Colon 26/IL-10 cells were respectively inoculated in different flanks of the same immunocompetent mice. This enhanced growth of wild-type tumors was not observed in nude mice. Immunocompetent mice that had rejected IL-2- or IL-12-secreting Colon 26 cells developed protective immunity and became completely resistant to wild-type Colon 26 cells subsequently challenged. However, some of the mice that had rejected IL-2 or IL-12 producers developed Colon 26/IL-10 tumors inoculated thereafter. The present study showed that production of IL-10 from tumor cells impaired T cell- and non-T cell-mediated systemic antitumor immunity in hosts.
Cancer Gene Therapy 02/2002; 9(1):109-15. DOI:10.1038/sj.cgt.7700418 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.
The Journal of Immunology 01/2001; 165(12):6816-24. DOI:10.4049/jimmunol.165.12.6816 · 4.92 Impact Factor