ジャーナル: EMBO Rep / 年: 2023 タイトル: CryoET shows cofilactin filaments inside the microtubule lumen. 著者: Camilla Ventura Santos / Stephen L Rogers / Andrew P Carter / 要旨: Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been ...Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
ダウンロード / ファイル: emd_18475.map.gz / 形式: CCP4 / 大きさ: 2.1 GB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Cryo-electron tomogram of an induced S2 cell protrusion after treatment with 2.5uM CytD (2h) and 2um thapsigargin (5h).
ボクセルのサイズ
X=Y=Z: 10.74 Å
密度
最小 - 最大
-0.60988736 - 0.43933672
平均 (標準偏差)
0.000000000000655 (±0.0399006)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
1440
1022
376
Spacing
1022
1440
376
セル
A: 10976.279 Å / B: 15465.6 Å / C: 4038.24 Å α=β=γ: 90.0 °
-
添付データ
-
試料の構成要素
-
全体 : Cytochalasin D-induced protrusion of a Drosophila S2 cell.
全体
名称: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
要素
細胞: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
-
超分子 #1: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
超分子
名称: Cytochalasin D-induced protrusion of a Drosophila S2 cell. タイプ: cell / ID: 1 / 親要素: 0 詳細: Cells were treated with 2uM thapsigargin for 5h and with 2.5uM Cytochalasin D for 2h prior to vitrification.
モデル: Quantifoil R3.5/1 / 材質: GOLD / メッシュ: 200 / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 30 sec. 詳細: Grids were glow discharged for 30s at 20mA and then coated with 0.25ug/mL Concanavalin A. Grids were washed twice with PBS before plating cells.
凍結
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 298 K / 装置: FEI VITROBOT MARK III
切片作成
その他: NO SECTIONING
位置合わせマーカー
Manufacturer: BBI Solutions / 直径: 10 nm
-
電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 平均露光時間: 0.56 sec. / 平均電子線量: 2.9 e/Å2