Feasibility of imaging intracellular tension probes in multicellular aggregates
Feasibility of imaging intracellular tension probes in multicellular aggregates
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多细胞聚集体中细胞内张力探针成像的可行性
DOI：
10.1117/12.2655401
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Boustany, Nada N.
中科院分区：
文献类型：
--
作者：
Hu, Luni;Ong, Julian;Barroso, Margarida M.;Boustany, Nada N.
多细胞聚集体构成了重要的三维（3D）模型，用于研究与肿瘤生长和进展相关的细胞行为，包括增殖和迁移。研究指出聚集体的生物力学环境在调节细胞增殖、分离和迁移中的作用。为了获得在细胞水平上的机械响应的测量，我们研究了使用分子张力探针在多细胞聚集体。虽然这些张力探针已用于二维单层细胞，但其应用于三维多细胞聚集体的可行性尚未得到证实。在本文中，我们利用先前描述的，频域荧光寿命显微镜（FLIM），测试的可行性，测量FRET使用的张力传感器（tenoculin TS），在CHO-K1细胞的多细胞聚集体。我们的数据表明，探针可以在球状体中表达，并且我们可以测量来自细胞内弥散分布的校准构建体的FLIM-FRET信号。当细胞表达白藜芦醇TS时，我们能够辨别荧光分布的变化，与校准构建体相比，点状染色的证据表明在粘附位点的定位。荧光寿命的斑点表达的tagulin TS是2.46 +/- 0.12 ns，长于2.21 +/- 0.103 ns，斑点表达无尾tagulin TS（VinTL），其中缺乏肌动蛋白结合结构域的寿命。与未加载VinTL对照相比，更长的寿命与跨黏着斑TS的更高张力一致。目前正在开展工作，以充分确定这些斑点的特征。
Multicellular aggregates constitute important 3-dimenstional (3D) models for investigating cellular behaviors, including proliferation and migration, that are relevant to neoplastic growth and progression. Studies point to the role of the aggregates’ biomechanical environment in modulating cell proliferation, segregation, and migration. To obtain measurements of mechanical responses at the cellular level, we investigate the use of molecular tension probes in multicellular aggregates. While these tension probes have been used in 2-dimensional monolayers of cells, the feasibility of their application in 3D multicellular aggregates has not been demonstrated yet. In this paper, we utilize a previously described, frequency-domain fluorescence lifetime microscope (FLIM), to test the feasibility of measuring FRET using the Vinculin Tension Sensor (Vinculin TS), in multicellular aggregates of CHO-K1 cells. Our data suggest that the probes can be expressed in the spheroids and that we can measure FLIM-FRET signal from calibration constructs that are diffusely distributed within the cells. When the cells express Vinculin TS, we were able to discern changes in fluorescence distribution, compared with the calibration constructs, with evidence of punctate staining suggestive of localization at adhesion sites. The fluorescence lifetime of puncta expressing Vinculin TS was 2.46 +/- 0.12 ns, longer than 2.21 +/- 0.103 ns, the lifetime of puncta expressing Tail-less Vinculin TS (VinTL), which lacks the actin-binding domain. The longer lifetime is consistent with higher tension across vinculin TS compared with the unloaded VinTL control. Work is underway to fully characterize these puncta.
影响因子：
3.5
作者：
Dumas, John-Paul;Jiang, James Y.;Boustany, Nada N.
通讯作者：
Boustany, Nada N.
FRET 结构频域荧光寿命成像中相寿命的线性行为
影响因子：
3.1
作者：
Sumetsky, Daniel;Jiang, James Y.;Ayad, Marina A.;Mahon, Timothy;Menaesse, Audrey;Cararo-Lopes, Marina M.;Patel, Mihir V.;Firestein, Bonnie L.;Boustany, Nada N.
通讯作者：
Boustany, Nada N.