Cell cycle dependent TN‐C promoter activity determined by live cell imaging

Abstract

The extracellular matrix protein tenascin-C plays a critical role in development, wound healing, and cancer progression, but how it is controlled and how it exerts its physiological responses remain unclear. By quantifying the behavior of live cells with phase contrast and fluorescence microscopy, the dynamic regulation of TN-C promoter activity is examined. We employ an NIH 3T3 cell line stably transfected with the TN-C promoter ligated to the gene sequence for destabilized green fluorescent protein (GFP). Fully automated image analysis routines, validated by comparison with data derived from manual segmentation and tracking of single cells, are used to quantify changes in the cellular GFP in hundreds of individual cells throughout their cell cycle during live cell imaging experiments lasting 62 h. We find that individual cells vary substantially in their expression patterns over the cell cycle, but that on average TN-C promoter activity increases during the last 40% of the cell cycle. We also find that the increase in promoter activity is proportional to the activity earlier in the cell cycle. This work illustrates the application of live cell microscopy and automated image analysis of a promoter-driven GFP reporter cell line to identify subtle gene regulatory mechanisms that are difficult to uncover using population averaged measurements. Published 2011 Wiley-Liss, Inc. y

Predicting rates of cell state change caused by stochastic fluctuations using a data-driven landscape model

“…Independent analysis of live-cell data in an unsorted dish confirmed that the dimmest cells (corresponding to the lower log normal in Fig. S2A) proliferated at a rate that was 10% slower than the other cells (31). This observation is consistent with previous observations that expression of TN-C is associated with proliferating cells (28).…”

“…Time-lapse fluorescence microscopy of live cells, in which individual cells were segmented and tracked over long times (31), reveals that TN-C promoter activity exhibits random fluctuations as well as cell cycle-dependent trends. On average, cells approximately double their total fluorescence before dividing.…”

“…The preparation of the cell line and all cell culture, imaging, and image analysis were performed as previously published (31). NIH 3T3 mouse fibroblasts (ATCC) were transfected with a construct of a 4.1-kbp fragment of the TN-C promoter (provided by Peter L. Jones, University of Pennsylvania, State College, PA) fused to a destabilized EGFP (Clontech Laboratories).…”

Automatic segmentation and supervised learning‐based selection of nuclei in cancer tissue images

“…The first measured the overlap between test and control segmentations using area similarity (AS) (49)(50)(51)(52) …”

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“…Live cell imaging allows examination of spatial and temporal information about gene expression in colonies and between colonies (Singer et al, 2014; Bajcsy et al, 2016; Halter et al, 2011; Barbaric et al, 2014; Chan et al, 2009). Meaningful quantification requires sampling a large population of cells and colonies, and because the area of individual colonies can be larger than the FOV with a 10× objective lens, tracking a colony over time as it grows and moves requires that many adjacent fields be examined and stitched together.…”