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Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1.

Sakata D, Taniguchi H, Yasuda S, Adachi-Morishima A, Hamazaki Y, Nakayama R, Miki T, Minato N, Narumiya S - J. Exp. Med. (2007)

Bottom Line: Although much is known about the latter, the physiological functions of mDia proteins are unclear.Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus.These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology,Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.

ABSTRACT
Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

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Related in: MedlinePlus

Decreased number of T cells in secondary lymphoid organs of mDia1−/− mice. (A) Western blot analysis. The homogenates of the brain, lung, and thymus of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were subjected to immunoblot analysis for mDia1, mDia2, mDia3, and β-actin (B) Weight of the body and lymphoid organs of wild-type (shaded bars) and mDia1−/− (open bars) mice (n = 3 for each group). The experiment was performed twice with reproducible results. (C) Immunohistochemistry of the spleen and lymph node. The spleen and lymph node of mDia1+/+ and mDia1−/− mice were stained for Thy1.2 (green) and B220 (red). Representative observation from samples of two mice. Bar, 100 μm. (D) Cell population analysis in the spleen, lymph node, and blood. The numbers of total cells, Thy1.2+ T cells, B220+ B cells, CD4+ T cells, CD8+ T cells, CD11c+ cells, and CD11b+ cells in the spleen, lymph node, and blood were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. (E) Cell population analysis in the thymus. The numbers of total cells and indicated subsets of thymocytes (DN, CD4−CD8−; DP, CD4+CD8+; CD4-SP, CD4+CD8−; CD8-SP, CD4−CD8+) in the thymus were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. The bottom panel shows the numbers of CD69loCD62LhiCD4-SP and CD8-SP cells in the thymus of mDia1+/+ and mDia1−/− mice (n = 4 for each group). Unless otherwise stated, experiments shown in D and E were performed using three mice for each group more than twice with similar results, and results from one experiment are shown. All data are shown as means ± SEM. *, P < 0.05 versus the number of the corresponding population in mDia1+/+ wild-type mice. The axillary and inguinal lymph nodes were used as peripheral lymph nodes for analysis in these experiments.
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fig1: Decreased number of T cells in secondary lymphoid organs of mDia1−/− mice. (A) Western blot analysis. The homogenates of the brain, lung, and thymus of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were subjected to immunoblot analysis for mDia1, mDia2, mDia3, and β-actin (B) Weight of the body and lymphoid organs of wild-type (shaded bars) and mDia1−/− (open bars) mice (n = 3 for each group). The experiment was performed twice with reproducible results. (C) Immunohistochemistry of the spleen and lymph node. The spleen and lymph node of mDia1+/+ and mDia1−/− mice were stained for Thy1.2 (green) and B220 (red). Representative observation from samples of two mice. Bar, 100 μm. (D) Cell population analysis in the spleen, lymph node, and blood. The numbers of total cells, Thy1.2+ T cells, B220+ B cells, CD4+ T cells, CD8+ T cells, CD11c+ cells, and CD11b+ cells in the spleen, lymph node, and blood were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. (E) Cell population analysis in the thymus. The numbers of total cells and indicated subsets of thymocytes (DN, CD4−CD8−; DP, CD4+CD8+; CD4-SP, CD4+CD8−; CD8-SP, CD4−CD8+) in the thymus were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. The bottom panel shows the numbers of CD69loCD62LhiCD4-SP and CD8-SP cells in the thymus of mDia1+/+ and mDia1−/− mice (n = 4 for each group). Unless otherwise stated, experiments shown in D and E were performed using three mice for each group more than twice with similar results, and results from one experiment are shown. All data are shown as means ± SEM. *, P < 0.05 versus the number of the corresponding population in mDia1+/+ wild-type mice. The axillary and inguinal lymph nodes were used as peripheral lymph nodes for analysis in these experiments.

Mentions: We generated an mDia1-floxed strain of mice that allows deletion of the translation initiation exon, Exon 1, of mDia1 on Cre/loxP-mediated recombination (Fig. S1, A and B, available at http://www.jem.org/cgi/content/full/jem.20062647/DC1). mDia1+/flox heterozygotes were then crossed with EIIa-Cre mice, in which Cre recombinase was expressed in the early embryo (12), to produce heterozygous mDia1+/− mice, which were intercrossed to produce homozygous mDia1−/− mice (Fig. S1 C). No expression of mDia1 protein was found in mDia1−/− mice, whereas expression of mDia2 and mDia3 proteins was not altered (Fig. 1 A). mDia1−/− mice were born with an expected Mendelian ratio (Fig. S1 D). Both male and female mDia1−/− mice developed without apparent abnormality and were fertile. Generated mDia1−/− mice were backcrossed for more than five generations to a C57BL/6 background and used for analysis with wild-type littermates obtained from the same heterozygous mating as a control. 8–12-wk-old mice were used in subsequent analyses.


Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1.

Sakata D, Taniguchi H, Yasuda S, Adachi-Morishima A, Hamazaki Y, Nakayama R, Miki T, Minato N, Narumiya S - J. Exp. Med. (2007)

Decreased number of T cells in secondary lymphoid organs of mDia1−/− mice. (A) Western blot analysis. The homogenates of the brain, lung, and thymus of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were subjected to immunoblot analysis for mDia1, mDia2, mDia3, and β-actin (B) Weight of the body and lymphoid organs of wild-type (shaded bars) and mDia1−/− (open bars) mice (n = 3 for each group). The experiment was performed twice with reproducible results. (C) Immunohistochemistry of the spleen and lymph node. The spleen and lymph node of mDia1+/+ and mDia1−/− mice were stained for Thy1.2 (green) and B220 (red). Representative observation from samples of two mice. Bar, 100 μm. (D) Cell population analysis in the spleen, lymph node, and blood. The numbers of total cells, Thy1.2+ T cells, B220+ B cells, CD4+ T cells, CD8+ T cells, CD11c+ cells, and CD11b+ cells in the spleen, lymph node, and blood were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. (E) Cell population analysis in the thymus. The numbers of total cells and indicated subsets of thymocytes (DN, CD4−CD8−; DP, CD4+CD8+; CD4-SP, CD4+CD8−; CD8-SP, CD4−CD8+) in the thymus were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. The bottom panel shows the numbers of CD69loCD62LhiCD4-SP and CD8-SP cells in the thymus of mDia1+/+ and mDia1−/− mice (n = 4 for each group). Unless otherwise stated, experiments shown in D and E were performed using three mice for each group more than twice with similar results, and results from one experiment are shown. All data are shown as means ± SEM. *, P < 0.05 versus the number of the corresponding population in mDia1+/+ wild-type mice. The axillary and inguinal lymph nodes were used as peripheral lymph nodes for analysis in these experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig1: Decreased number of T cells in secondary lymphoid organs of mDia1−/− mice. (A) Western blot analysis. The homogenates of the brain, lung, and thymus of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were subjected to immunoblot analysis for mDia1, mDia2, mDia3, and β-actin (B) Weight of the body and lymphoid organs of wild-type (shaded bars) and mDia1−/− (open bars) mice (n = 3 for each group). The experiment was performed twice with reproducible results. (C) Immunohistochemistry of the spleen and lymph node. The spleen and lymph node of mDia1+/+ and mDia1−/− mice were stained for Thy1.2 (green) and B220 (red). Representative observation from samples of two mice. Bar, 100 μm. (D) Cell population analysis in the spleen, lymph node, and blood. The numbers of total cells, Thy1.2+ T cells, B220+ B cells, CD4+ T cells, CD8+ T cells, CD11c+ cells, and CD11b+ cells in the spleen, lymph node, and blood were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. (E) Cell population analysis in the thymus. The numbers of total cells and indicated subsets of thymocytes (DN, CD4−CD8−; DP, CD4+CD8+; CD4-SP, CD4+CD8−; CD8-SP, CD4−CD8+) in the thymus were determined in mDia1+/+ (shaded bars) and mDia1−/− (open bars) mice. The bottom panel shows the numbers of CD69loCD62LhiCD4-SP and CD8-SP cells in the thymus of mDia1+/+ and mDia1−/− mice (n = 4 for each group). Unless otherwise stated, experiments shown in D and E were performed using three mice for each group more than twice with similar results, and results from one experiment are shown. All data are shown as means ± SEM. *, P < 0.05 versus the number of the corresponding population in mDia1+/+ wild-type mice. The axillary and inguinal lymph nodes were used as peripheral lymph nodes for analysis in these experiments.
Mentions: We generated an mDia1-floxed strain of mice that allows deletion of the translation initiation exon, Exon 1, of mDia1 on Cre/loxP-mediated recombination (Fig. S1, A and B, available at http://www.jem.org/cgi/content/full/jem.20062647/DC1). mDia1+/flox heterozygotes were then crossed with EIIa-Cre mice, in which Cre recombinase was expressed in the early embryo (12), to produce heterozygous mDia1+/− mice, which were intercrossed to produce homozygous mDia1−/− mice (Fig. S1 C). No expression of mDia1 protein was found in mDia1−/− mice, whereas expression of mDia2 and mDia3 proteins was not altered (Fig. 1 A). mDia1−/− mice were born with an expected Mendelian ratio (Fig. S1 D). Both male and female mDia1−/− mice developed without apparent abnormality and were fertile. Generated mDia1−/− mice were backcrossed for more than five generations to a C57BL/6 background and used for analysis with wild-type littermates obtained from the same heterozygous mating as a control. 8–12-wk-old mice were used in subsequent analyses.

Bottom Line: Although much is known about the latter, the physiological functions of mDia proteins are unclear.Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus.These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology,Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan.

ABSTRACT
Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

Show MeSH
Related in: MedlinePlus