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Fluorescent images for the dynamic behaviors of inner nuclear membrane proteins and NLS-BSA during in vitro nuclear envelope reconstitution reaction

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posted on 2024-06-03, 09:56 authored by Tomoko Funakoshi, Naoko Imamoto

Fluorescent images of LBR-YFP, CFP-emerin and Cy3-NLS-BSA during the nuclear envelope reconstitution reaction in mitotic semi-intact cells. 

Semi-intact cells prepared from HeLa cells stably co-expressing LBR-Venus and SECFP-emerin by digitonin treatment. Images were captured just after the addition of a reaction mixture to digitonin-permeabilized semi-intact cells and at 5-min intervals throughout the reactions for 60 min (t01~t013) at 30 °C with a DeltaVision microscope. The reaction mixture was composed of an ATP regeneration system, GTP, mitotic cytosol, cyclin-dependent kinase 1 inhibitor (RO-3306) and Cy3 labeled bovine serum albumin tagged with SV40 T-antigen nuclear localization signal (Cy3-NLS-BSA) as a transport indicator. 

The file names indicate fluorescent labeled proteins (LBR-Venus , LBR-YFP; SECFP-emerin, CFP-emerin; Cy3-NLS-BSA), mitotic phases (metaphase, anaphase, telophase) of semi-intact cells, and the incubation time point (t01~t13) of each image. The merged images (merge) show images of SECFP-emerin (blue), LBR-Venus (green) and Cy3-NLS-BSA (red).

Funding

Analysis of function of Hikeshi affecting cellular homeostasis

Japan Society for the Promotion of Science

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Division of cellular function of nuclear transport pathways

Japan Society for the Promotion of Science

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History

Corresponding author email address

n-imamoto@juhs.ac.jp

Title (in Japanese)

in vitro核膜再構築反応における核膜内膜タンパク質と核タンパク質(NLS-BSA)の動態

Description (in Japanese)

分裂期セミインタクトHeLa細胞における核膜再構成反応中のLBR-YFP、CFP-emerinおよびCy3-NLS-BSAの動態を蛍光画像で示す。 LBR-VenusとSECFP-emerinを安定に発現させたHeLa細胞をジギトニンで処理してセミインタクト細胞を調整した。セミインタクト細胞に反応混合液を加え、30℃で60分間(t01~t013)反応させた。反応混合液を加えた直後から5分間隔でDeltaVision顕微鏡により画像撮影した。反応混合物は、ATP再生系、GTP、分裂期HeLa細胞から調整した細胞質、CDK1阻害剤(RO-3306)の混合液に、Cy3-NLS-BSA(SV40 T抗原の核局在シグナルを付加したCy3標識ウシ血清アルブミン)を核輸送マーカーとして加えた。 ファイル名は、各画像のタンパク質名(LBR-Venus, LBR-YFP; SECFP-emerin, CFP-emerin; Cy3-NLS-BSA)、セミインタクト細胞を調整した細胞の分裂期各ステージ(metaphase、anaphase、telophase)、反応時間(t01~t13)を示す。マージ画像(merge)はSECFP-emerin(青)、LBR-Venus(緑)、Cy3-NLS-BSA(赤)を示す。

Manuscript title (in Japanese)

核膜サブドメイン形成を指標にした分裂期セミインタクト細胞を用いた核膜再構成解析系の樹立

Authors (in Japanese)

船越智子, 今本尚子

Copyright

© 2024 The Author(s)

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