Sviatlana Shashkova

Single-molecule fluorescence microscopy review: shedding new light on old problems

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

Molecular communication: crosstalk between the Snf1 and other signaling pathways

The yeast Saccharomyces cerevisiae employs different conserved signaling pathways to adapt to altered availability of nutrient and energy sources. Crosstalk between the pathways occurs to integrate different internal and external stimuli and adjust cellular metabolism, growth and proliferation to altered environmental conditions. The main glucose repression pathway, Snf1/Mig1, plays an essential role in adaptation to glucose limitation. However, the Snf1 protein kinase is also involved in regulation of many other cellular processes. We summarize evidence that Snf1 is part of a network of communicating pathways, and we suggest research directions that may help elucidating signal flow within this network.

Transcription factor clusters regulate genes in eukaryotic cells

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms

A yeast Saccharomyces cerevisiae Snf1 kinase, an analog of mammalian AMPK, regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

Network reconstruction and validation of the Snf1/AMPK pathway in baker’s yeast based on a comprehensive literature review

Background/Objectives:The SNF1/AMPK protein kinase has a central role in energy homeostasis in eukaryotic cells. It is activated by energy depletion and stimulates processes leading to the production of ATP while it downregulates ATP-consuming processes. The yeast SNF1 complex is best known for its role in glucose derepression.Methods:We performed a network reconstruction of the Snf1 pathway based on a comprehensive literature review. The network was formalised in the rxncon language, and we used the rxncon toolbox for model validation and gap filling.Results:We present a machine-readable network definition that summarises the mechanistic knowledge of the Snf1 pathway. Furthermore, we used the known input/output relationships in the network to identify and fill gaps in the information transfer through the pathway, to produce a functional network model. Finally, we convert the functional network model into a rule-based model as a proof-of-principle.Conclusions:The workflow presented here enables large scale reconstruction, validation and gap filling of signal transduction networks. It is analogous to but distinct from that established for metabolic networks. We demonstrate the workflow capabilities, and the direct link between the reconstruction and dynamic modelling, with the Snf1 network. This network is a distillation of the knowledge from all previous publications on the Snf1/AMPK pathway. The network is a knowledge resource for modellers and experimentalists alike, and a template for similar efforts in higher eukaryotes. Finally, we envisage the workflow as an instrumental tool for reconstruction of large signalling networks across Eukaryota.