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Abstract：
Differences in gene-expression profiles between individual cells can give rise to distinct cell fate decisions. Yet how localisation on a micropattern impacts initial changes in mRNA, protein, and phosphoprotein abundance remains unclear. To identify the effect of cellular position on gene expression, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridisation (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins, and phosphoproteins in single cells. We studied 67 (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns. ARTseq-FISH reveals relative changes in both abundance and localisation of mRNAs and (phospho-)proteins during the first 48 hours of exit from pluripotency. We confirm these changes by conventional immunofluorescence and time-lapse microscopy. Chemical labelling, immunofluorescence, and single-cell time-lapse microscopy further show that cells closer to the edge of the micropattern exhibit increased proliferation compared to cells at the centre. Together these data suggest that while gene expression is still highly heterogeneous position-dependent differences in mRNA and protein levels emerge as early as 12 hours after LIF withdrawal.
摘要：
单个细胞之间基因表达谱的差异可以引起不同的细胞命运决定。然而，微图案上的定位如何影响mRNA的初始变化，蛋白质，磷蛋白丰度尚不清楚。为了确定细胞位置对基因表达的影响，我们开发了一种可扩展的抗体和mRNA靶向顺序荧光原位杂交（ARTseq-FISH）方法，能够同时分析mRNA，蛋白质，和单个细胞中的磷蛋白。我们研究了在圆形微图案上培养的单个小鼠胚胎干细胞（mESC）中的67个（磷酸）蛋白和mRNA靶标。ARTseq-FISH揭示了在离开多能性的前48小时期间mRNA和(磷酸)蛋白的丰度和定位的相对变化。我们通过常规免疫荧光和延时显微镜证实了这些变化。化学标签,免疫荧光，和单细胞延时显微镜进一步显示，与中心细胞相比，更靠近微图案边缘的细胞增殖增加。这些数据一起表明，尽管基因表达仍然是高度异质的，但mRNA和蛋白质水平的位置依赖性差异早在LIF戒断后12小时就出现。
