ジャーナル: Dev Cell / 年: 2019 タイトル: Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures. 著者: Patrick C Hoffmann / Tanmay A M Bharat / Michael R Wozny / Jerome Boulanger / Elizabeth A Miller / Wanda Kukulski / 要旨: Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and ...Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways.
ダウンロード / ファイル: emd_10301.map.gz / 形式: CCP4 / 大きさ: 816 MB / タイプ: IMAGE STORED AS SIGNED BYTE
注釈
CLEM of resin-embedded GFP-Ist2 scs2/22 tcb1/2/3 deletion mutant yeast cell shown in Figure 2F of publication
ボクセルのサイズ
X=Y=Z: 11.02 Å
密度
最小 - 最大
-128 - 127
平均 (標準偏差)
46.536835 (±3.915299)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
-102
サイズ
2048
2048
204
Spacing
2048
2048
204
セル
A: 22568.96 Å / B: 22568.96 Å / C: 2248.08 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
envelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z
11.020000488281
11.020000488281
11.02
M x/y/z
2048
2048
204
origin x/y/z
0.000
0.000
0.000
length x/y/z
22568.961
22568.961
2248.080
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
111
-94
150
NX/NY/NZ
111
123
111
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
-102
NC/NR/NS
2048
2048
204
D min/max/mean
-128.000
127.000
46.537
-
添付データ
-
試料の構成要素
-
全体 : Correlative FM and ET of resin-embedded scs2/22 tcb1/2/3 deletion...
全体
名称: Correlative FM and ET of resin-embedded scs2/22 tcb1/2/3 deletion mutant yeast cell expressing GFP-Ist2
要素
細胞: Correlative FM and ET of resin-embedded scs2/22 tcb1/2/3 deletion mutant yeast cell expressing GFP-Ist2
-
超分子 #1: Correlative FM and ET of resin-embedded scs2/22 tcb1/2/3 deletion...
超分子
名称: Correlative FM and ET of resin-embedded scs2/22 tcb1/2/3 deletion mutant yeast cell expressing GFP-Ist2 タイプ: cell / ID: 1 / 親要素: 0
由来(天然)
生物種: Saccharomyces cerevisiae (パン酵母)
-
実験情報
-
構造解析
手法
ネガティブ染色法, クライオ電子顕微鏡法
解析
電子線トモグラフィー法
試料の集合状態
cell
-
試料調製
緩衝液
pH: 7.4
染色
タイプ: NONE / 材質: Uranyl Acetate 詳細: 0.03-0.05 % uranyl acetate during freeze substitution and post-staining of sections with Reynold's lead citrate
糖包埋
材質: Lowicryl HM20
凍結
凍結剤: NITROGEN
加圧凍結法
装置: OTHER 詳細: The value given for _emd_high_pressure_freezing.instrument is Leica EM HPM100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION ...詳細: The value given for _emd_high_pressure_freezing.instrument is Leica EM HPM100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
切片作成
ウルトラミクロトーム - 装置: Reichert Ultracut E ウルトラミクロトーム - 温度: 298 K / ウルトラミクロトーム - 最終 厚さ: 270 nm
位置合わせマーカー
Manufacturer
直径
Electron Microscopy Sciences
15 nm
Life Technologies
50 nm
-
電子顕微鏡法
顕微鏡
FEI TECNAI F20
詳細
STEM mode on an axial brightfield detector with a high-tilt tomography holder (Fischione Instruments, Model 2020)
撮影
フィルム・検出器のモデル: OTHER / 平均電子線量: 1000.0 e/Å2 詳細: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder at 1.102 nm pixel size with a camera length of 200 mm. [NOTE: Electron dose unknown. Value specified here is a dummy]
電子線
加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: OTHER / 撮影モード: BRIGHT FIELD
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
-
画像解析
最終 再構成
ソフトウェア - 名称: eTomo / 詳細: 239 tilted images used from two axes / 使用した粒子像数: 239