An integrated image analysis platform to quantify signal transduction in single cells.

Microscopy can provide invaluable information about biological processes at the single cell level. It remains a challenge, however, to extract quantitative information from these types of datasets. We have developed an image analysis platform named YeastQuant to simplify data extraction by offering an integrated method to turn time-lapse movies into single cell measurements. This platform is based on a database with a graphical user interface where the users can describe their experiments. The database is connected to the engineering software Matlab, which allows extracting the desired information by automatically segmenting and quantifying the microscopy images. We implemented three different segmentation methods that recognize individual cells under different conditions, and integrated image analysis protocols that allow measuring and analyzing distinct cellular readouts. To illustrate the power and versatility of YeastQuant, we investigated dynamic signal transduction processes in yeast. First, we quantified the expression of fluorescent reporters induced by osmotic stress to study noise in gene expression. Second, we analyzed the dynamic relocation of endogenous proteins from the cytoplasm to the cell nucleus, which provides a fast measure of pathway activity. These examples demonstrate that YeastQuant provides a versatile and expandable database and an experimental framework that improves image analysis and quantification of diverse microscopy-based readouts. Such dynamic single cell measurements are highly needed to establish mathematical models of signal transduction pathways.

[1]  C. Pesce,et al.  Regulated cell-to-cell variation in a cell-fate decision system , 2005, Nature.

[2]  Megan N. McClean,et al.  Signal processing by the HOG MAP kinase pathway , 2008, Proceedings of the National Academy of Sciences.

[3]  B. Reid,et al.  Chromophore formation in green fluorescent protein. , 1997, Biochemistry.

[4]  B Hamilton,et al.  Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. , 1998, Genes & development.

[5]  Glòria Mas,et al.  Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes , 2009, The EMBO journal.

[6]  Dana H. Ballard,et al.  Generalizing the Hough transform to detect arbitrary shapes , 1981, Pattern Recognit..

[7]  Ali Kinkhabwala,et al.  Regulation of Ras Localization by Acylation Enables a Mode of Intracellular Signal Propagation , 2010, Science Signaling.

[8]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[9]  A. Turjanski,et al.  MAP kinases and the control of nuclear events , 2007, Oncogene.

[10]  J E Ferrell,et al.  The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. , 1998, Science.

[11]  Fabian Rudolf,et al.  Transient Activation of the HOG MAPK Pathway Regulates Bimodal Gene Expression , 2011, Science.

[12]  R. Yu,et al.  Fus3 generates negative feedback that improves information transmission in yeast pheromone response , 2008, Nature.

[13]  M. Elowitz,et al.  Frequency-modulated nuclear localization bursts coordinate gene regulation , 2008, Nature.

[14]  Jerome T. Mettetal,et al.  The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisiae , 2008, Science.

[15]  Gustav Ammerer,et al.  Acute glucose starvation activates the nuclear localization signal of a stress‐specific yeast transcription factor , 2002, The EMBO journal.

[16]  Wendell A Lim,et al.  A microfluidic system for dynamic yeast cell imaging. , 2008, BioTechniques.

[17]  Gaudenz Danuser,et al.  Computer Vision in Cell Biology , 2011, Cell.

[18]  Martha S. Cyert,et al.  Regulation of Nuclear Localization during Signaling* , 2001, The Journal of Biological Chemistry.

[19]  Matthias Peter,et al.  MAP kinase dynamics in response to pheromones in budding yeast , 2001, Nature Cell Biology.

[20]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[21]  Gilles Charvin,et al.  A Microfluidic Device for Temporally Controlled Gene Expression and Long-Term Fluorescent Imaging in Unperturbed Dividing Yeast Cells , 2008, PloS one.

[22]  P. Swain,et al.  Stochastic Gene Expression in a Single Cell , 2002, Science.

[23]  David E. Levin,et al.  Cell Wall Integrity Signaling in Saccharomyces cerevisiae , 2005, Microbiology and Molecular Biology Reviews.

[24]  R. Durbin,et al.  Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes , 2010, Nature.

[25]  G. Ammerer,et al.  Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[26]  U. Jung,et al.  The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. , 1995, Genes & development.

[27]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.