Durrieu Lucía, Bush Alan, Grande Alicia, Johansson Rikard, Janzén David, Katz Andrea, Cedersund Gunnar, Colman-Lerner Alejandro
Department of Physiology, Molecular and Cellular Biology, School of Exact and Natural Sciences, University of Buenos Aires (UBA), C1428EGA, Argentina.
Institute of Physiology, Molecular Biology and Neurosciences, National Council of Scientific and Technical Research (IFIBYNE-UBA-CONICET), C1428EGA, Argentina.
iScience. 2022 Dec 29;26(1):105906. doi: 10.1016/j.isci.2022.105906. eCollection 2023 Jan 20.
Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive transport, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.
核转运是真核细胞功能的重要组成部分。在此，我们展示了单细胞荧光漂白后恢复（scFRAP），这是一种基于模型辅助的光漂白后荧光恢复（FRAP）方法，用于在单个活细胞中独立测定核输入和输出速率。为了克服单细胞测量中固有的噪声，我们在同一个细胞上进行了连续的FRAP实验。我们发现同基因酵母群体中转运速率存在很大的细胞间差异。对于被动转运，核孔复合体数量的变异性可能解释了大部分变异性。利用这种方法，我们研究了转录因子Ace2在活跃核穿梭过程中的母细胞-子细胞不对称性，Ace2在G1早期特异性地集中在子细胞核中。与之前假设的子细胞输出减少不同，我们发现这种不对称主要是由于子细胞中输入增加。这些结果揭示了细胞动力学中的细胞间差异及其来源。
