Periodic Reporting for period 4 - GLYCONOISE (Emergent properties of cell surface glycosylation in cell-cell communication) Reporting period: 2020-09-01 to 2022-02-28 Summary of the context and overall objectives of the project Every living cell is decorated with a dense fur of glycans. Multicellular organisms make use of this matrix to encode for specific information such as cellular identity, metabolic and activation status, and circadian clock. Cell surface glycans, present on glycoproteins and glycolipids modulate protein localization, their interaction partners and receptor life cycles. Since glycans are secondary gene products and their occurrence and modulation cannot be predicted from analysis of the genome per se, their formation as well as their breakdown is determined by many factors, not limited to gene expression of potentially over 200 enzymes and transporters. Regulatory mechanisms of this fine-tuned interplay arose on many different time scales: very quick alteration of the glycocalyx can be introduced using secreted hydrolases and endo- and exocytosis of glycoproteins. Long-term remodeling of glycans can result from gene expression and histone modifications. How can such a specially and temporally regulated, complex and stochastic system encode reliable for information that can successfully be decoded by other cells through the use of lectin receptors? Here we address the fundaments of how cell surface exposed sugars in multicellular organisms serve as information storage and how they are decoded. For this we treat this system like a communication channel in which a sender cell conveys a defined message to a receiver cell. This formalism opens the door for cross-disciplinary approaches coming from theoretical physics (i.e. information theory) to aid the analysis of the biological data. A central figure in any communication channel is noise and we will assess the different layers of noise and dissect their origin and influence. We combine insights from atomic resolution understanding the biophysics of protein glycan interaction with experimental assessment of cellular mechanisms addressing these questions: How often does a protein-carbohydrate interaction on a cell surface lead to a productive, biological response? How do lectins and glycans find each other on two-dimensional surfaces? How much information can be transferred through such a communication channel and how does this compare to glycan-independent pathways of cell-cell communication? Taken together these insights will help us to understand why almost all tumor cells change their cell surface glycans in a similar way. Moreover, we gain insight into fundamental processes in biology that lead to better understanding of how glycosylation became such an essential posttranslational modification present in every living organism. Furthermore, nature uses sugar for specific delivery of cargo to certain cells in the body and with our knowledge on how such system works, we can address molecular drug targeting strategies more efficiently to reduce side effects of novel therapeutics. Finally, our insights might stimulate the design of novel communication channels based on heteromultivalent low affinity interactions. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have established a cellular reporter system with which we can monitor the information flow from incoming glycans (free, present on proteins or scaffold) and they receptors. This is highly modular and we can alter the input in various ways, as well as the reporter system. We setup a pipeline to analyse complex biological data using customized scripts, which now yield a quantitative picture of the cellular communication channel. This enables the analysis of the two emerging properties of the biological channel: robustness and noise. We find that sugar-based communication has a higher intrinsic noise regardless of the lower affinity of the input. The reasons are not fully understood but likely are associated with lectin and glycan redundancy. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Using the model system for glycan-based cell-cell communication, we can now quantify the information flow between a sender and a receiver cell using information theory. This was not possible beforehand and opens the door to a systematic evaluation of the underlying processes. The system is modular invites other disciplines to add insights coming from genomics and metabolomics. Moreover, an approach has been developed, which allows us to investigate several hundred incoming particles on the cell surface during single particle tracking analysis. In such a high density of particles, an analysis was impossible. With our technology, including newly developed code, particle fusion and splitting can be monitored in terabytes of life cell imaging data. We are also addressing the problem of single cell glycome analysis. Important key experiments have been performed and we are now approaching a solution of this longstanding problem in glycobiology in the remaining funding period.