イノシトールリン脂質4位脱リン酸化酵素TMEM55Aは食飽に局在することでマクロファージの貪食を抑制的に制御する

概要

༤ኈㄽᩥ

࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁ 4 ఩⬺ࣜࣥ㓟໬㓝⣲
TMEM55A ࡣ㣗⬊࡟ᒁᅾࡍࡿࡇ࡜࡛
࣐ࢡࣟࣇ࢓࣮ࢪࡢ㈎㣗ࢆᢚไⓗ࡟ไᚚࡍࡿ

ᗈᓥ኱Ꮫ኱Ꮫ㝔་ṑ⸆ಖ೺Ꮫ◊✲⛉
༤ኈㄢ⛬ ་ṑ⸆Ꮫᑓᨷ
๰⸆ᶆⓗศᏊ⛉Ꮫ◊✲ᐊ

D170096  ᳃ᒸ ┿

┠ḟ
ᗎㄽ…………………………………………………………………………………………………...1
1 ᐇ㦂᪉ἲ…………………………………………………………………………………………...6
1-1 TMEM55A/TMEM55B Ⓨ⌧ᢚไ⣽⬊ࡢస〇………………………………………….6
1-2 ⣽⬊ᇵ㣴…………………………………………………………………………………...6
1-3 IgG ឤస㉥⾑⌫ࡢㄪ〇࡜

51Cr

ࢆ⏝࠸ࡓ㈎㣗άᛶࡢ ᐃ……………………………...8

1-4 ⺯ග㢧ᚤ㙾࡟ࡼࡿ IgG ឤస㉥⾑⌫㈎㣗άᛶࡢ ᐃ…………………………………..8
1-5 zymosan ㈎㣗άᛶࡢ ᐃ……………………………………………………………......8
1-6 ኱⭠⳦㈎㣗άᛶࡢ ᐃ……………………………………………………………………...9
1-7 RAW264.7 ⣽⬊࡬ࡢ PtdIns(5)P ᑟධ……………………………………………………..9
1-8 ࣉࣛࢫ࣑ࢻ…………………………………………………………………………….....10
1-9 ࢺࣛࣥࢫࣇ࢙ࢡࢺ…………………………………………………………………….....10
1-10 ㈎㣗㐣⛬࡟࠾ࡅࡿ࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁ࠊF-actin ࡢືែほᐹ…………………....10
1-11 3xHis-3xPHD ࢆ⏝࠸ࡓ PtdIns(5)P ᐃ㔞…………………………………………...12
1-12 ࢚࢘ࢫࢱࣥࣈࣟࢵࢸ࢕ࣥࢢ………………………………………………………......13
1-13 LC-MS/MS ࢩࢫࢸ࣒࡟ࡼࡿ PtdIns(3,4,5)P3 ࡢᐃ㔞………….………………......13
1-14 ⤫ィゎᯒ………………………………………………………………………………......13
2 ⤖ᯝ……………………………………………………………………………………….……...11
2-1 TMEM55A/TMEM55B Ⓨ⌧ᢚไ⣽⬊ࡢస〇………………………………………......14
2-2 shTMEM55A ⣽⬊࡛ࡣ኱ࡁ࠸⢏Ꮚࡢ㈎㣗άᛶࡀஹ㐍ࡍࡿ………………………........17
2-3 TMEM55A/TMEM55B ࡣ㣗⬊࡟ᒁᅾࡍࡿ…………………………………………......23
  2-4PtdIns(4,5)P2 ࡢ⵳✚࡟ࡼࡗ࡚㈎㣗ࡀஹ㐍ࡍࡿ……………………………………........29
2-5 shTMEM55A ⣽⬊࡛ࡣ IgG ่⃭᫬࡟ PtdIns(3,4,5)P3 ࡀቑຍࡍࡿ……………........33
2-6 TMEM55A ࡣ㣗⬊⭷ୖࡢ PIPs ືែ࡟㛵୚ࡍࡿ………………………..………........36
2-7 shTMEM55A ⣽⬊࡛ࡣ F-actin ࡢ㔜ྜ-⬺㔜ྜࢧ࢖ࢡࣝࡀಁ㐍ࡉࢀࡿ…..…..…........43
3 ⪃ᐹ……………………………………………………………………………………………….45
⥲ᣓ………………………………………………………………………………………………….46
ཎⴭㄽᩥ…………………………………………………………………………………………….48
ཧ⪃ᩥ⊩…………………………………………………………………………………………….49
ㅰ㎡………………………………………………………………………………………………….53

ᗎㄽ
 ࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁㸦PIPs㸧ࡣぶỈᇶ࡜ࡋ࡚࢖ࣀࢩࢺ࣮ࣝ⎔ࢆ᭷ࡍࡿࢢࣜࢭࣟࣜࣥ⬡
㉁࡛࠶ࡿࠋࡇࡢ࢖ࣀࢩࢺ࣮ࣝࡢ 3 ఩ࠊ4 ఩ࠊ5 ఩ࡀྍ㏫ⓗ࡟ࣜࣥ㓟໬ಟ㣭ࡉࢀࡿࡇ࡜࡛ࠊ
PIPs ࡟ࡣ 8 ✀㢮ࡀᏑᅾࡍࡿ㸦Fig. 1㸧
ࠋ
ࡇࡢࣜࣥ㓟໬≧ែࡣ 19 ✀㢮ࡢࣜࣥ㓟໬㓝⣲㸦kinase㸧
࡜ 29 ✀㢮ࡢ⬺ࣜࣥ㓟໬㓝⣲㸦phosphatase㸧࡟ࡼࡗ࡚┦஫࡟ኚ᥮ࡉࢀࡿࠋPIPs ࡣᏑᅾ㔞ⓗ
࡟ࡣࣜࣥ⬡㉁඲యࡢ 10%࡟‶ࡓࡎࠊࡑࡢ௦ㅰᅇ㌿ࡣ㏿࠸ࠋࡑࡢࡓࡵࠊ⣽⬊⭷ࡢᵓ⠏ᮦᩱ࡜
ࡋ࡚௨እࡢᶵ⬟ࢆᣢࡘࡇ࡜ࡀྂࡃ࠿ࡽᣦ᦬ࡉࢀ࡚࠸ࡓࠋPIPs ࡣࡑࢀࡒࢀ࡟≉␗ⓗ࡞ᶆⓗࢱ
ࣥࣃࢡ㉁࡜⤖ྜࡋࠊࡑࡢάᛶࡸ⣽⬊ෆᒁᅾࢆไᚚࡍࡿࡇ࡜࡛ࢩࢢࢼࣝఏ㐩ࢆࡣࡌࡵࠊᵝࠎ࡞
⣽⬊ᶵ⬟ࢆไᚚࡋ࡚࠸ࡿࠋ⚾ࡣࡇࢀࡲ࡛ PIPs ࡜ PIPs ௦ㅰ㓝⣲ࡢ⏕⌮ᶵ⬟ࡸ⑌ᝈ࡜ࡢ࠿࠿
ࢃࡾ࡟ࡘ࠸࡚◊✲ࢆ㐍ࡵ࡚ࡁࡓࠋ
 ࣐ࢡࣟࣇ࢓࣮ࢪࡣ⑓ཎ⳦ࡸ⮬ᕫࡢṚ⣽⬊ࠊኚᛶ≀㉁࡞࡝ࢆ㈎㣗ࡋࠊᾘ໬ࡍࡿࡇ࡜࡛⏕࿨ࡢ
ᜏᖖᛶ⥔ᣢ࡟㈉⊩ࡍࡿ[1]-[3]ࠋ࣐ࢡࣟࣇ࢓࣮ࢪࡀ␗≀ࢆㄆ㆑ࡍࡿ࡜ࠊ␗≀ࡣ⣽⬊⭷ࡢఙᒎ࡟
ࡼࡗ࡚ᤕࡽ࠼ࡽࢀࠊ⥆࠸࡚⭷ࡢ㛢㙐࡟ࡼࡗ࡚㣗⬊࡟ᑒධࡉࢀࡿࠋ㈎㣗ࡢヲ⣽࡞࣓࢝ࢽࢬ࣒ࡣ
㛵୚ࡍࡿཷᐜయࡸᶆⓗ࡜ࡍࡿ⢏Ꮚࡢࢧ࢖ࢬ࡟౫Ꮡࡋ࡚ኚ໬ࡍࡿࠋࡓࡔࡋ PIPs ࢆ㏻ࡌࡓไᚚ
ࢆཷࡅࡿ࡜࠸࠺Ⅼ࡟ࡘ࠸࡚ࡣඹ㏻ࡋ࡚࠸ࡿࠋ౛࠼ࡤࠊF-actin ࡢ⵳✚ࡣ PtdIns(4,5)P2 ࡟ࡼ
ࡗ࡚ಁ㐍ࡉࢀࠊ௬㊊ࡢఙᒎࢆಁࡍࠋࡇࡢ㐣⛬ࡣ㈎㣗ࡍࡿᶆⓗࡢ✀㢮࡟㛵ࢃࡽࡎᶆⓗࢆ᭱ึ࡟
ᤕࡲ࠼ࡿࡓࡵ࡟ᚲ㡲࡛࠶ࡿ[4]-[6]ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⥆ࡃ phagocytic cup ࡀ㛢⎔ࡍࡿẁ㝵࡛
ࡣᶆⓗࡀ኱ࡁ࠸ሙྜ㸦>4 Pm㸧࡜ᑠࡉ࠸ሙྜࡣูࡢ࣓࢝ࢽࢬ࣒࡛㐍⾜ࡍࡿࠋࢡࣛࢫ I ࡢ PI3K
ࡀ PtdIns(4,5)P2 ࢆࣜࣥ㓟໬ࡍࡿࡇ࡜୍࡛㐣ᛶ࡟㣗⬊࡟⵳✚ࡍࡿ PtdIns(3,4,5)P3 ࡣࠊ኱ࡁ
࡞⢏Ꮚࢆྲྀࡾ㎸ࡴ㝿࡟ᚲ㡲࡜࡞ࡿ㸦Fig. 2㸧ࠋࡋ࠿ࡋࡇࡢ㐣⛬ࡣᑠࡉ࡞⢏Ꮚࢆྲྀࡾ㎸ࡴሙྜ
࡟ࡣ୙せ࡛࠶ࡿ [7]-[9]ࠋ
PtdIns(3,4,5)P3 ࡣ phagocytic cup ࡟ RhoGAP ࢆࣜࢡ࣮ࣝࢺࡍࡿࠋ
ࡑ࠺ࡍࡿࡇ࡜࡛ Rho ࣇ࢓࣑࣮ࣜGTPase ࡢ୙άᛶ໬࡜࢔ࢡࢳࣥ㔜ྜࡢ⤊⤖ࢆᘬࡁ㉳ࡇࡍ࡜
ሗ࿌ࡉࢀ࡚࠸ࡿ[9]ࠋࡇࡢࡼ࠺࡞ F-actin ࡢ༠ㄪⓗ࡞⬺㔜ྜࡀᕧ኱࡞ phagocytic cup ࡢ㛢㙐
࡟ࡣᚲせ࡛࠶ࡿ[10]ࠋ
 TMEM55a ཬࡧ TMEM55b㸦Transmembrane protein 55A/B㸧ࡣ့ங㢮࡛᭱ึ࡟ྠᐃࡉ
ࢀࡓ PtdIns(4,5)P2 ࡢ 4 ఩⬺ࣜࣥ㓟໬㓝⣲࡛࠶ࡿ[11]ࠋࡇࢀࡽ஧ࡘࡢ㓝⣲ࡣ⬺ࣜࣥ㓟໬㓝⣲
ࡢࢥࣥࢭࣥࢧࢫ㓄ิ㸦CX5R motif㸧࡜᥎ᐃୖࡢ 2 ᅇ⭷㈏㏻㡿ᇦࢆ᭷ࡋ࡚࠸ࡿ㸦Fig. 3㸧
ࠋࡇ
ࢀࡽࡢ㓝⣲άᛶ࡜ᇶ㉁≉␗ᛶࡣ in vitro ุ࡛ᐃࡉࢀࡓࡶࡢ࡛࠶ࡾࠊin vivo ࡟࠾ࡅࡿ⬺ࣜࣥ
㓟໬㓝⣲࡟ࡼࡿ PIPs ࡢ PtdIns(5)P ࡬ࡢኚ᥮ࡣᮍࡔ᳨ウࡀ㐍ࢇ࡛࠸࡞࠸ࠋTMEM55a ࡜
TMEM55b ࢆᇵ㣴⣽⬊࡟㐣๫Ⓨ⌧ࡉࡏࡓሙྜࠊࡇࢀࡽࡣ࡜ࡶ࡟ᚋᮇ࢚ࣥࢻࢯ࣮࣒/ࣜࢯࢯ࣮
࣒⭷࡟ᒁᅾࡋࠊࣜ࢞ࣥࢻ⤖ྜᚋࡢ EGF ཷᐜయࡢศゎࢆಁ㐍ࡍࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ[11]ࠋࡋ
࠿ࡋ࡞ࡀࡽࠊTMEM55a/TMEM55b ࡜ྠࡌ㓝⣲άᛶࢆ᭷ࡍࡿ⣽⳦⏤᮶⑓ཎᛶᅉᏊࡢ IpgD



ࢆᇵ㣴⣽⬊࡟Ⓨ⌧ࡉࡏࡓሙྜࠊEGF ཷᐜయࡢศゎࡣᢚไࡉࢀࡿ[12]-[14]ࠋࡇࡢࡼ࠺࡟ࠊ
PtdIns(4,5)P2 ࡢ 4 ఩⬺ࣜࣥ㓟໬㓝⣲ࡢ⏕⌮ᶵ⬟࡟ࡘ࠸࡚ࡣ኱㒊ศࡀ㐣๫Ⓨ⌧ᐇ㦂࡟ࡼࡿ᥎
 ࡛࠶ࡿࠋTMEM55B ࡣ⣽⬊᰾ࡢ PtdIns(5)P ࢆ⏘⏕ࡍࡿࡇ࡜࡛ࠊp53 ࡢάᛶ໬ࢆ㏻ࡌࡓ࢔
࣏ࢺ࣮ࢩࢫࡢಁ㐍࡟㛵୚ࡍࡿ࡜࠸ࡗࡓሗ࿌ࡀᏑᅾࡍࡿࡶࡢࡢ[15]ࠊTMEM55A ࡟ࡘ࠸࡚ࡣ
⣽⬊ෆ࡛ࡢᙺ๭࡟ࡘ࠸࡚ゎᯒࡋࡓ◊✲ࡀᏑᅾࡏࡎࠊࡑࡢ⏕⌮ⓗᶵ⬟ࡣ୙᫂░࡞㒊ศࡀከ࠸
ࡢࡀ⌧≧࡛࠶ࡿࠋ
 ࣐ࢡࣟࣇ࢓࣮ࢪࡣ๓㏙ࡢࡼ࠺࡟㈎㣗㐣⛬࡟࠾࠸࡚ PIPs ࡢไᚚࢆཷࡅ࡚࠸ࡿ࡯࠿[7]-[9]ࠊ
㈎㣗ᚋࡢᾘ໬㐣⛬[16]-[19]ࡸࢧ࢖ࢺ࢝࢖ࣥ⏘⏕[20]-[21]ࡢไᚚ࡟࠾࠸࡚ࡶ PIPs ࡀ㔜せ࡞ᙺ
๭ࢆᯝࡓࡋ࡚࠸ࡿࠋච␿ᶵ⬟ࡔࡅ࡛࡞ࡃࠊ࣐ࢡࣟࣇ࢓࣮ࢪࡢ୺せ࡞ᙺ๭ࡢࡦ࡜ࡘ࡛࠶ࡿኚᛶ
ࢥࣞࢫࢸ࣮ࣟࣝࡢฎ⌮࡟࠾࠸࡚ࡶ PIPs ࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࢆ⚾ࡓࡕࡣሗ࿌ࡋ࡚࠸ࡿ[22]ࠋ
ࡇࡢࡼ࠺࡟࣐ࢡࣟࣇ࢓࣮ࢪᶵ⬟࡟࠾࠸࡚ PIPs ࡢᯝࡓࡍᙺ๭ࡀ኱ࡁ࠸ࡇ࡜࠿ࡽࠊPIPs ௦ㅰ
㓝⣲ࡢᶵ⬟ࢆ᳨ウࡍࡿୖ࡛࣐ࢡࣟࣇ࢓࣮ࢪࡣ㠀ᖖ࡟᭷⏝࡛࠶ࡿࠋࡑࡇ࡛⚾ࡣ PIPs ௦ㅰ㓝⣲
ࡢ୰࡛ࡶ≉࡟ᶵ⬟ゎᯒࡀ㐍ࢇ࡛࠸࡞࠸ TMEM55A ࡟╔┠ࡋࠊ࣐ࢡࣟࣇ࢓࣮ࢪ⣔ᇵ㣴⣽⬊ᰴ
RAW264.7 ⣽⬊ࢆ฼⏝ࡋ࡚ TMEM55A ࡢ⏕⌮ⓗᶵ⬟ࢆ᫂ࡽ࠿࡟ࡍࡿ➃⥴ࢆ㛤ࡇ࠺࡜ヨࡳ
ࡓࠋ⚾ࡓࡕࡢ◊✲ᐊ࡛㛤Ⓨࡋࡓຠ⋡ⓗ࡞࢚ࣞࢡࢺ࣏࣮ࣟࣞࢩࣙࣥἲ࡟ࡼࡗ࡚ RAW264.7 ⣽
⬊࡟ shRNA ࢆᑟධࡋ࡚ TMEM55A Ⓨ⌧ᢚไ⣽⬊㸦௨ୗࠊshTMEM55A ⣽⬊㸧ࢆస〇ࡋ
ࡓࠋࡇࡢ⣽⬊ࢆ⏝࠸࡚࣐ࢡࣟࣇ࢓࣮ࢪࡢᶵ⬟ㄪ⠇࡟࠾ࡅࡿ TMEM55A ࡢᙺ๭ࡢゎ᫂࡟ྲྀࡾ
⤌ࢇࡔࠋᮏ◊✲࡛ࡣࠊࡇࢀࡲ࡛⣽⬊ෆ࡛ࡢാࡁࡀศ࠿ࡗ࡚࠸࡞࠿ࡗࡓ TMEM55A ࡀ࣐ࢡࣟ
ࣇ࢓࣮ࢪࡢ㈎㣗㐣⛬ࢆไᚚࡋ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ㸦Fig. 2㸧ࠋ



Fig. 1 ࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁ࡢᵓ㐀



Fig. 2 ㈎㣗㐣⛬ࡢ࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁



Fig. 3 TMEM55A/TMEM55B ࡢ࢔࣑ࣀ㓟㓄ิ
 NCBI Reference Sequence Database ࡼࡾࣄࢺ TMEM55A ࡜ TMEM55B ࡢ࢔࣑ࣀ
㓟㓄ิࢆྲྀᚓࡋࠊࡑࡢ㓄ิࢆẚ㍑ࡋࡓ㸦TMEM55A: NP_061180.1ࠊTMEM55B:
NP_653169.2㸧
ࠋ㢮ఝᛶࡢ㧗࠸࢔࣑ࣀ㓟ࢆ㯮ሬࡾ࡛♧ࡋ࡚࠾ࡾࠊ”3”ࡣỈ㓟ᇶࢆᣢࡘ࢔
࣑ࣀ㓟ࠊ”4”ࡣሷᇶᛶ࢔࣑ࣀ㓟ࠊ”5”ࡣⰾ㤶᪘࢔࣑ࣀ㓟ࠊ”6”ࡣ⬡⫫᪘࢔࣑ࣀ㓟ࢆࡑࢀࡒ
ࢀ♧ࡋ࡚࠸ࡿࠋ⥳Ⰽࡢᯟࡣ⬺ࣜࣥ㓟໬㓝⣲άᛶ㒊఩ࡢ CX5R motif ࢆ♧ࡋ࡚࠸ࡿࠋ㉥Ⰽ
ࡢᯟࡣ TMHMM Server v. 2.0 ࢆ౑⏝ࡋ࡚ண ࡋࡓ⭷㈏㏻㡿ᇦࢆ♧ࡋ࡚࠸ࡿࠋ



1 ᐇ㦂᪉ἲ
1-1 TMEM55A/TMEM55B Ⓨ⌧ᢚไ⣽⬊ࡢస〇
 TMEM55A ࡶࡋࡃࡣ TMEM55B ࢆⓎ⌧ᢚไࡋࡓ RAW264.7 ⣽⬊ࡣ௨ୗࡢ㏻ࡾస〇ࡋ
ࡓࠋ
 TMEM55AࠊTMEM55B ࢆᶆⓗ࡜ࡍࡿ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ pH1 ࣋ࢡࢱ࣮࡟ᑟධࡋࠊ
siRNA ࣊࢔ࣆࣥࢆⓎ⌧ࡉࡏࡓ㸦Table. 1㸧
ࠋᶆⓗ㓄ิࡢࡑࢀࡒࢀ࡟ࡘ࠸࡚ࠊḟࡢ㓄ิࢆ⏝࠸
୍࡚ᑐࡢ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆྜᡂࡋࡓࠋ5′-CCC(X)19TTCAAGAGA(Y)19TTTTTGGAAA3′ཬࡧ 5′-CTAGTTTCCAAAAA(Y)19TCTCTTGAA(X)19GGGTGCA-3′ࠊ(X)19 ࡣࢥ࣮ࢻ㓄
ิࠊ(Y)19 ࡣࡑࡢ┦⿵㓄ิ࡛࠶ࡿࠋ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ࢔ࢽ࣮ࣜࣥࢢࡋࠊpH1 ࣋ࢡࢱ࣮ࡢ
PstI ཬࡧ XbaI ࢧ࢖ࢺ࡛ H1 RNA ࣉ࣮ࣟࣔࢱ࣮ࡢୗὶ࡟⤖ྜࡉࡏࡓࠋ250 V/950 PF ࡛
5x106㹼1x107 cells ࡢ RAW264.7 ⣽⬊࡟ୖグ࣋ࢡࢱ࣮ࢆ㑇ఏᏊᑟධࡋࡓ㸦Gene Pulser II;
Bio-Rad㸧
ࠋ㑇ఏᏊᑟධࡢ 24 ᫬㛫ᚋࠊ⣽⬊࡟ 3 Pg/mL ࡢ puromycin ࢆῧຍࡋࠊ⣽⬊ࡢ㑅
ᢥࢆ⾜ࡗࡓࠋ㑇ఏᏊࡢⓎ⌧ຠ⋡ࢆㄪ࡭ࡿࡓࡵࠊSepasol ࡛ torla RNA ࢆᢳฟࡋࠊ௨ୗࡢࣉ
ࣛ࢖࣐࣮ࢆ⏝࠸࡚ᐃ㔞ࣜ࢔ࣝࢱ࢖࣒ PCR ࡛ mRNA ࢆᐃ㔞ࡋࡓ㸦Table. 2㸧
ࠋᮏ◊✲࡛ࡣ
mRNA Ⓨ⌧ࢆ 60~80%⛬ᗘ࡟ᢚไࡉࢀࡓࢡ࣮ࣟࣥࢆ㑅ᢥࡋࡓࠋControl ⣽⬊ࡣᶆⓗ㓄ิࡢ
௦ࢃࡾ࡟ 400 bp ࡢࢫࢱࢵࣇ࢓࣮㓄ิࢆྵࢇࡔ pH1 ࣋ࢡࢱ࣮ࢆ⏝࠸࡚స〇ࡋࡓࠋ
1-2 ⣽⬊ᇵ㣴
 ࣇ࢓ࢦࢧ࢖ࢺ࣮ࢩࢫ࢔ࢵࢭ࢖ࡸ㢧ᚤ㙾ほᐹ࡛ࡣࠊ⣽⬊ࢆ 24-well plate ࡸ tisue culturecoated glass bottom dish ࡟᧛✀ࡋࠊ4.5 g/L ࢢࣝࢥ࣮ࢫࠊ10% FBS ࢆྵࡴ RPMI1640 ᇵ
ᆅ୰࡛ຍ‵ୗ 5% CO2ࠊ37Υ࡛ᇵ㣴ࡋࡓࠋᐇ㦂㛤ጞ๓࡟ᇵᆅࢆ㝖ཤࡋࠊNaHCO3 ࢆྵࡲ
ࡎࠊ20 mM HEPES/NaOH ࢆῧຍࡋࡓ RPMI1640 ᇵᆅ࡟஺᥮ࡋࡓࠋ37ΥࡢỈᾎୖࠊ኱Ẽ
୰࡛✀ࠎࡢάᛶ ᐃࢆ⾜ࡗࡓࠋ



TMEM55A 229

5̓-GTGCACAGTTTGCAATGAA-3̓

TMEM55A 322

5̓-GGATACATCTCGGCGAATA-3̓

TMEM55B

5̓-GCATCAGCATGTAGTCAAA-3̓

Table. 1 shRNA ࡢᶆⓗ㓄ิ

TMEM55A (F)

5̓-CGGGAAATGTCACTCCCACA-3̓

TMEM55A (R)

-CFFGTTCCTGGACTGGCGAT-3̓

TMEM55B (F)

5̓-CCGCCCCAGGCATTTCC-3̓

TMEM55B (R)

5̓-TATCCGGGCTAGTCAAGGGT-3̓

Table. 2 ᐃ㔞ࣜ࢔ࣝࢱ࢖࣒ PCR ⏝ࣉࣛ࢖࣐࣮



1-3 IgG ឤస㉥⾑⌫ࡢㄪ〇࡜ 51Cr ࢆ⏝࠸ࡓ㈎㣗άᛶࡢ ᐃ
 ㉥⾑⌫㸦RBC㸧ࡣ 51Cr ࡛ᶆ㆑ࡋࡓ[23]ࠋᶆ㆑㉥⾑⌫ࢆᢠ⨺㉥⾑⌫ᢠయ࡜࡜ࡶ࡟ࠊ0.1%
gelatinࠊ75 mM NaClࠊ0.15 mM CaCl2ࠊ0.5 mM MgCl2ࠊ10 mM EDTA ࢆྵࢇࡔ 5 mM
veronal buffer㸦pH7.5㸧
㸦GVB㸧୰࡛ 37Υࠊ10 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ
 IgG ឤస㉥⾑⌫ࡢ⤖ྜ㔞࡜㈎㣗㔞ࡣඛࡢᩥ⊩࡟ᚑࡗ࡚ ᐃࡋࡓ[23]ࠋ༢ᒙࡢ RAW264.7
⣽⬊㸦24-well plate ࡛ 2x105 cells/well㸧ࢆ 51Cr ᶆ㆑ IgG ឤస㉥⾑⌫࡜࡜ࡶ࡟ᅗ࡟♧ࡋࡓ
᫬㛫 37Υ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋࠊPBS(-)࡛ 3 ᅇὙίࡋࠊᮍ⤖ྜࡢ IgG ឤస㉥⾑
⌫ࢆ㝖ཤࡋࡓᚋࠊ0.1 mL ࡢపᙇ PBS(-)࡟▷᫬㛫᭚㟢ࡋࡓࠋᚓࡽࢀࡓୖΎࡢᨺᑕάᛶࢆ࣐
ࢡࣟࣇ࢓࣮ࢪࡢ⾲㠃࡟⤖ྜࡋࡓ IgG ឤస㉥⾑⌫ࡢ⤖ྜ㔞࡜ࡋ࡚ ᐃࡋࡓࠋ⣽⬊ࢆ෌ᗘ
PBS(-)࡛ 3 ᅇὙίࡋࠊ0.5% Triton X-100 ࡛ḷㅴ໬ࡋࡓࠋ⁐ᾮ୰ࡢᨺᑕάᛶࢆྲྀࡾ㎸ࡲࢀ
ࡓ IgG ឤస㉥⾑⌫ࡢ㔞࡜ࡋ࡚ ᐃࡋࡓࠋ
1-4 ⺯ග㢧ᚤ㙾࡟ࡼࡿ IgG ឤస㉥⾑⌫㈎㣗άᛶࡢ ᐃ
 FLAG ࢱࢢࡸ EGFP ࡟ࡼࡗ࡚ᶆ㆑ࡉࢀࡓྛࢥࣥࢫࢺࣛࢡࢺࢆࢺࣛࣥࢫࣇ࢙ࢡࢺࡋࡓ
RAW264.7 ⣽⬊ࢆ tissue culture-coated glass bottom dish ࡟᧛✀ࡋࠊ5% CO2ࠊ37Υ࡛
4~6 ᫬㛫ᇵ㣴ࡋࡓࠋ↓ᶆ㆑ࡢ IgG ឤస㉥⾑⌫ࢆຍ࠼࡚⣽⬊ࢆịୖ࡛ 15 ศ㛫࢖࣮ࣥ࢟ࣗ࣋
ࢺࡋࡓᚋࠊࡉࡽ࡟ 37Υ࡛ 15 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋࠊPBS(-)࡛ 3 ᅇὙίࡋࠊᮍ
⤖ྜࡢ IgG ឤస㉥⾑⌫ࢆ㝖ཤࡋࡓᚋࠊ4% paraformaldehyde/PBS(-)ࢆ⏝࠸࡚ᐊ ࡛ 15 ศ
㛫⣽⬊ࢆᅛᐃࡋࡓࠋ0.1% saponin/3% BSA/TBS ࡛ 60 ศ㛫ᢞୗฎ⌮ࢆ⾜࠸ࠊAlexa Fluor
647 ⤖ྜᆺࡢᢠ࢘ࢧࢠ IgG ᢠయࢆ⏝࠸ࡓච␿ᰁⰍ࡟ࡼࡗ࡚㉥⾑⌫ࡢྲྀࡾ㎸ࡳ㔞ࢆホ౯ࡋ
ࡓࠋFLAG ࢱࢢ࡟ࡘ࠸࡚ࡣᢠ FLAG ᢠయ㸦1:1000ࠊSigmaࠊ#F3165㸧࡜ Alexa Fluor 488
⤖ྜᆺᢠ࣐࢘ࢫ IgG ஧ḟᢠయ㸦Cell Signaling㸧ࢆ⏝࠸࡚ච␿ᰁⰍࡋࡓࠋྲྀࡾ㎸ࢇࡔ㉥⾑
⌫ᩘࡢᐃ㔞࡟ࡣ BZ-H2C analysis system㸦Keyence㸧ࢆ౑⏝ࡋࡓࠋ
1-5 zymosan ㈎㣗άᛶࡢ ᐃ
 ィᩘࢆ⡆౽໬ࡍࡿࡓࡵࠊFITC ᶆ㆑ zymosan ࢆྠ㔞ࡢ㠀ᶆ㆑ zymosan ࡜ΰྜࡋࠊ⣙ 1
ศ㛫㉸㡢Ἴฎ⌮ࡋࡓࠋRAW264.7 ⣽⬊㸦105 cells/wellࠊglass bottom dish㸧࡟ᑐࡋ࡚
2x106 ಶࡢ zymosan ࢆຍ࠼࡚ 37Υ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ㈎㣗㐣⛬ࡣị෭ࡋࡓ PBS(-)ࢆ
ῧຍࡍࡿࡇ࡜࡛೵Ṇࡉࡏࡓࠋࡑࡢᚋ PBS(-)࡛ 3 ᅇὙίࡋࠊ4% paraformaldehyde/PBS(-)
ࢆ⏝࠸࡚ᐊ ࡛ 15 ศ㛫⣽⬊ࢆᅛᐃࡋࡓࠋ↓సⅭ࡟㑅ࢇࡔ 3 ど㔝࠿ࡽ 100 ಶ௨ୖࡢ⣽⬊ࡢ
⺯ග⏬ീ㸦ບ㉳Ἴ㛗 488 nmࠊ⺯ගἼ㛗 525 nm㸧࡜఩┦ᕪ⏬ീࢆ⺯ග㢧ᚤ㙾࡛᧜ᙳࡋࡓࠋ
Control ⣽⬊ࡀྲྀࡾ㎸ࢇࡔ zymozan 㔞ࢆ”1”࡜ࡋ࡚ྛ⣽⬊ࡀྲྀࡾ㎸ࢇࡔ zymosan 㔞ࡢᖹᆒ
್ࢆࢢࣛࣇ࡟♧ࡋࡓࠋ



1-6 ኱⭠⳦㈎㣗άᛶࡢ ᐃ
 ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࢆ⏝࠸ࡓ PCR ࡟ࡼࡗ࡚ pmCherry-N1 ࣋ࢡࢱ࣮㸦Clontechࠊ
Mountain View㸧ࢆ㗪ᆺ࡜ࡋ࡚ mCherry ࡢ㓄ิࢆቑᖜࡋࠊࡉࡽ࡟ 5’ᮎ➃࡜ 3’ᮎ➃࡟ࡑࢀ
ࡒࢀ BamHI ࡜ EcoRI ࢧ࢖ࢺࢆᑟධࡋࡓࠋቑᖜ⏘≀ࢆ pGEX-4T-3 ࣋ࢡࢱ࣮࡟ࢡ࣮ࣟࢽࣥ
ࢢࡋ࡚స〇ࡋࡓ࣋ࢡࢱ࣮ࢆ BL-21 ኱⭠⳦࡟ࢺࣛࣥࢫࣇ࢛࣮࣒ࡋࡓࠋmCherry ࡢㄏᑟࡣ௨
ୗࡢࡼ࠺࡟⾜ࡗࡓࠋࢩࣥࢢࣝࢥࣟࢽ࣮ࢆ 10 mL ࡢ Luria–Bertani㸦LB㸧ᇵᆅ୰࡛ࠊ37Υ
୍࡛ᬌ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ኱⭠⳦ࢆ 100 mL ࡢ LB ᇵᆅ࡟ᗈࡆࠊOD600 ࡀ 0.8 ࡟㐩ࡍࡿ
ࡲ࡛ 37Υ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋ0.5 mM ࡢ isopropyl-β-d-thiogalactopyranoside
㸦IPTG㸧ࢆῧຍࡋࠊ30Υ࡛ 4 ᫬㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡍࡿࡇ࡜࡛ࢱࣥࣃࢡ㉁ࡢⓎ⌧ࢆㄏᑟࡋ
ࡓࠋmCherry Ⓨ⌧኱⭠⳦ࢆ PBS ࡛Ὑίࡋࠊ4% paraformaldehyde/PBS(-)ࢆ⏝࠸࡚ᐊ ࡛
15 ศ㛫ᅛᐃࡋࡓࠋᶆ㆑኱⭠⳦ࢆᢠ኱⭠⳦ᢠయࢆ⏝࠸࡚ GVB ୰࡛ឤసࡋࠊὙίࡋࡓᚋ౑⏝
๓࡟ 3 ศ㛫㉸㡢Ἴฎ⌮ࡋࡓࠋglass bottom dish ࡟᧛✀ࡋࡓ RAW264.7 ⣽⬊࡟ᶆ㆑኱⭠⳦
ࢆ 1:50 ࡢ๭ྜ࡛ῧຍࡋ࡚ịୖ࡛ 20 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋࡉࡽ࡟ 37Υ࡛ 20 ศ
㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚㈎㣗ࡉࡏࡓࠋ㈎㣗㐣⛬ࡣị෭ࡋࡓ pH5.5 ࡢ RPMI1640 ᇵᆅ࡛ 3 ᅇ
Ὑίࡍࡿࡇ࡜࡛೵Ṇࡉࡏࠊ⣽⬊⾲㠃ࡢ኱⭠⳦ࢆࡣࡀࡋࡓࠋ4% paraformaldehyde/PBS(-)ࢆ
⏝࠸࡚ᐊ ࡛ 15 ศ㛫⣽⬊ࢆᅛᐃࡋࡓࠋ↓సⅭ࡟㑅ࢇࡔ 3 ど㔝࠿ࡽ 100 ಶ௨ୖࡢ⣽⬊ࡢ⺯
ග⏬ീ㸦ບ㉳Ἴ㛗 587 nmࠊ⺯ගἼ㛗 610 nm㸧࡜఩┦ᕪ⏬ീࢆ⺯ග㢧ᚤ㙾࡛᧜ᙳࡋࡓࠋ
Control ⣽⬊ࡀྲྀࡾ㎸ࢇࡔ mCherry ᶆ㆑኱⭠⳦㔞ࢆ”1”࡜ࡋ࡚ྛ⣽⬊ࡀྲྀࡾ㎸ࢇࡔ
mCherry ᶆ㆑኱⭠⳦㔞ࡢᖹᆒ್ࢆࢢࣛࣇ࡟♧ࡋࡓࠋ
1-7 RAW264.7 ⣽⬊࡬ࡢ PtdIns(5)P ᑟධ
 C16 ࡢ PtdIns(5)P ࢆ CHCl3:MeOH:H2O㸦1:2:0.8㸧ࡢΰྜ⁐፹࡟⁐ゎࡋ࡚ࠊ-80Υ࡛ಖ
ᏑࡋࡓࠋPtdIns(5)P ⁐ᾮࢆ❅⣲࢞ࢫ࡛஝⇱ࡉࡏࠊ࢟ࣕࣜ࢔࣮ࢱࣥࣃࢡ㉁࡜ 1:1 ࡢ mol ẚ࡛
ΰྜࡋࡓࠋΰྜ⁐ᾮࢆ 2 ศ㛫㉸㡢Ἴฎ⌮ࡋࠊᐊ ࡛ 10 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋㄪ〇ࡋ
ࡓ PIPs-࢟ࣕࣜ࢔࣮ࢱࣥࣃࢡ㉁」ྜయࢆ RPMI1640 ᇵᆅ࡟ᕼ㔘ࡋ࡚⣽⬊࡟ῧຍࡋࡓ[24]ࠋ



1-8 ࣉࣛࢫ࣑ࢻ
 myosin X ࡢࢱࣥࢹ࣒ࢻ࣓࢖ࣥ PH1(N)-PH2-PH3(C)㸦Myo-PH㸧ࢆ࣐࢘ࢫ࣐ࢡࣟࣇ࢓࣮
ࢪࡼࡾ௨ୗࡢࣉࣛ࢖࣐࣮ࢆ⏝࠸࡚༢㞳ࡋࡓ㸦5’ -AGATCTCCCTATTTCCACAGTTTTC3’ࠊ5’ - GAATTCCTACTTGGATCTCTGCAGCA-3’㸧
ࠋࡘ࠸࡛ࡇࡢࢻ࣓࢖ࣥࢆ pEGFP-C1
ࡢ BglII ࡜ EcoRI ࢧ࢖ࢺ࡟ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࡓࠋpEGFP-C1-IpgD㸦㔝⏕ᆺ㸦WT㸧ཬࡧ
⬺ࣜࣥ㓟໬㓝⣲άᛶḞᦆኚ␗య㸦PD㸧
㸧
ࠊpEGFP-N1-PLCδ-PH ཬࡧ pcDNA3.1 ࡟ࢡ࣮ࣟ
ࢽࣥࢢࡉࢀࡓ TMEM55A ࡜ TMEM55BࠊPIPK IIDࡣబࠎᮌ㞝ᙪᩍᤵ㸦ᮾி་⛉ṑ⛉኱
Ꮫ㸧ࡼࡾࡈᥦ౪࠸ࡓࡔ࠸ࡓࠋTMEM55A ࡜ TMEM55B ࡟ࡘ࠸࡚ࡣ pmcherry-C1 ࣋ࢡࢱ
࣮࡟ EcoRI ࡜ BamHI ࢧ࢖ࢺ࡛ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࡓࠋshRNA ࡟ᑐࡍࡿⓎ⌧ᅇ᚟ᐇ㦂ࡢ
ࡓࡵ࡟ࠊTMEM55A ࡢ㓄ิࢆᶆⓗ࡜ࡋ࡚ࢧ࢖ࣞࣥࢺኚ␗ࢆᑟධࡋࡓ㸦Table. 3㸧
ࠋPIPK
IID࡟ࡘ࠸࡚ࡣ pEGFP-C1 ࣋ࢡࢱ࣮࡟ KpnI ࡜ XbaI ࢧ࢖ࢺ࡛ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࡓࠋࡲ
ࡓࠊEGFP ࢱࢢ௜ࡅࡉࢀࡓ Rab5ࠊRab7ࠊRab11ࠊRab20 ࡣ⚟⏣ᩍᤵ㸦ᮾ໭኱Ꮫ㸧ࡼࡾࡈ
ᥦ౪࠸ࡓࡔ࠸ࡓ[25]ࠋEGFP ࢱࢢࡢ Lifeact ࡣ㐣ཤࡢሗ࿌࡟ᚑࡗ࡚⏝ពࡋࡓ[26]ࠋ
1-9 ࢺࣛࣥࢫࣇ࢙ࢡࢺ
 NeonTM transfection system㸦Invitorogen㸧ࢆ⏝࠸࡚ࢺࣛࣥࢫࣇ࢙ࢺࡋࡓࠋࡓࡔࡋࠊ
RAW264.7 ⣽⬊⏝࡟ᙜ◊✲ᐊ࡛㛤Ⓨࡋࡓ buffer㸦25 mM Hepesࠊ62.5 mM KClࠊ10 mM
K2HPO4ࠊ2 mM MgCl2ࠊ125 mM Treharoseࠊ0.5 % Ficoll㸧ࢆ⏝࠸ࡓࠋ᮲௳ࡶ᭱㐺໬ࢆ
⾜࠸ࠊ10 PL ࢳࢵࣉ࡟࡚ 1800 V x 20 ms x 1ࠊ2 ᅇ/well ࡜ࡋࡓࠋࢺࣛࣥࢫࣇ࢙ࢡࢺࡋࡓ⣽
⬊ࢆ glass bottom dish ࡟᧛✀ࡋࠊ20% FBS ࢆྵ᭷ࡋࡓ RPMI1640 ୰࡛ 5~24 ᫬㛫࢖ࣥ࢟
࣮ࣗ࣋ࢺࡋࡓᚋ㸦ᇵ㣴᫬㛫ࡣ౑⏝ࡋࡓࣉࣛࢫ࣑ࢻ࡟ࡼࡗ࡚␗࡞ࡿ㸧ࠊ⣽⬊ࢆ㢧ᚤ㙾ゎᯒ࡟
౑⏝ࡋࡓࠋ
1-10 ㈎㣗㐣⛬࡟࠾ࡅࡿ࢖ࣀࢩࢺ࣮ࣝࣜࣥ⬡㉁ࠊF-actin ࡢືែほᐹ
 ✀ࠎࡢ⺯ගࣉ࣮ࣟࣈ㸦Table. 4㸧ࢆࢺࣛࣥࢫࣇ࢙ࢡࢺࡋࡓ⣽⬊ࢆ glass bottom dish ࡟᧛
✀ࡋࠊIgG ឤస㉥⾑⌫ῧຍᚋࠊ37Υࡢ㢧ᚤ㙾ྎ࡟⛣ࡋࡓࠋCFI Plan Apo VC60 xH Ἔᾐࣞ
ࣥࢬࢆ⿦╔ࡋࡓ BIOREVO BZ9000 㢧ᚤ㙾㸦Keyence㸧࡛㈎㣗㐣⛬ࢆ᧜ᙳࡋࡓࠋ㣗⬊ࡢ⺯
ගࢆ BZ-II analysis system㸦Keyence㸧⏝࠸ࡓࠋ



TMEM55A (F)

5̓-GGTTAAGTGCACCGTGTGCAATGAAGCTAC-3̓

TMEM55A (R)

5̓-GTAGCTTCATTGCACACGGTGCACTTAACC-3̓

Table. 3 TMEM55A ኚ␗ᑟධ⏝ࣉࣛ࢖࣐࣮

Probe

Target

EGFP-MyoX-PH

PtdIns(3,4,5)P3

EGFP-PLCG-PH

PtdIns(4,5)P2

EGFP-Lifeact

F-actin

Table. ...

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