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Proteins are one of the key components of cellular life that play a crucial role in most biological processes. Therefore, quantification of protein copy numbers is essential for revealing and better understanding of cellular behavior and functions. Here we describe a single-molecule fluorescence-based method of protein copy number quantification directly in living cells. This enables quick and reliable estimations and comparison of the protein of interest abundance without implementing large-scale studies.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Combined pdf comprising 1 supplementary table, 15 supplementary figures, supplementary methods, supplementary movie legends, and supplementary refererences

Highlights • Single-molecule imaging to determine genome organization in live budding yeast. • Utilize astigmatism imaging to enable extraction of 3D position data. • Use a transcription factor Mig1 to mark the genome at target genes. • Map out 3D coordinates of target genes with millisecond sampling. • Observe differences to 3C structure indicating dynamic heterogeneity of the genome., How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living cells.

Aggregation of misfolded or damaged proteins is often attributed to numerous metabolic and neurodegenerative disorders. To reveal underlying mechanisms and cellular responses, it is crucial to investigate protein aggregate dynamics in cells. Here, we used super-resolution single-molecule microscopy to obtain biophysical characteristics of individual aggregates of a model misfolded protein ∆ssCPY* labelled with GFP. We demonstrated that oxidative and hyperosmotic stress lead to increased aggregate stoichiometries but not necessarily the total number of aggregates. Moreover, our data suggest the importance of the thioredoxin peroxidase Tsa1 for the controlled sequestering and clearance of aggregates upon both conditions. Our work provides novel insights into the understanding of the cellular response to stress via revealing the dynamical properties of stress-induced protein aggregates.

How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living 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 budding yeast Saccharomyces cerevisiae AMP-activated protein kinase/SNF1 is a member of a highly conserved protein family present in all eukaryotes. Snf1 regulates energy homeostasis; in yeast, it is best-known for its role in cellular adaptation to glucose limitation. Utilisation of carbon and energy sources other than glucose is controlled, among others, via regulation of gene expression. Expression of genes essential for metabolism of alternative sources, such as maltose, galactose and sucrose is regulated by the transcriptional repressor Mig1, which in turn is controlled by Snf1. Mig1 shuttles in and out of the nucleus in response to glucose availability, which makes it a convenient read-out for Snf1 pathway activity in single cell analysis. The overall goal of this thesis is to achieve a better understanding of the dynamic control of an AMPK/SNF1 signal transduction pathway using the budding yeast S. cerevisiae as a model system. We combined classic biochemical, molecular and microbiology approaches with cutting-edge biophysical and imaging methods to fill gaps in our understanding of signal transduction mechanisms. Being the first ones to use millisecond imaging to monitor signal transduction, thus, the first ones to observe single Mig1 molecules in live cells, we found that regardless of glucose availability Mig1 is present in the cytoplasm and the nucleus as monomer and oligomers. We observed similar clusters of the transcription activator Msn2. Thus, we suggest that eukaryotic gene regulation is mediated through transcription factors which act as multimeric clusters. The structure of those clusters is stabilised by depletion forces that mediate interactions between intrinsically disordered regions of transcription factors. Classic biochemical approaches revealed a dual mechanism of Mig1 dephosphorylation which includes glucose-dependent and glucose-independent events. We also found evidence for a novel step of Mig1 regulation which includes tyrosine phosphorylation. We show that the expression of Mig1 is itself glucose-regulated in a Snf1-dependent manner. Taken together, this work provides novel concepts in understanding of the AMPK/Snf1 signal transduction pathway with specific emphasis on Mig1 regulation.