Abstract
This thesis describes various projects focusing on the development of live-cell single-molecule imaging assays to grasp dynamic processes.
Chapter 2 and 3 focus on heterogeneity of translation on single mRNAs over time. Inspired by the SunTag translation imaging system, an orthogonal live-cell single-molecule nascnet polypeptide labeling system called MoonTag is developed
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in Chapter 2. The SunTag and MoonTag system can be combined to simultaneously analyze translation of multiple mRNA species in the same cell and to facilitate investigations into translation of multiple ORFs on the same mRNA. Additionally, yet another tag is developed - the MashTag - to interrogate canonical and non-canonical translation simultaneously on single mRNAs. Using the expanded single-molecule translation imaging toolbox, we observe widespread non-canonical translation due to alternative translation start site selection. Canonical and non-canonical translation start site selection is heterogenous between mRNA molecules of the same gene and can even be variable over time on the same mRNA molecule.
On single mRNAs, we frequently observed fluctuations in translation over time. To examine the contribution of ribosome recruitment to variations in translation, we analyze the dynamics of eIFs in Chapter 3. We uncover that binding of a single eIF4E molecule to an mRNA molecule can be responsible for translation initiation of multiple consecutive ribosomes. Furthermore, we speculate that the dynamics of eIF4E binding to a cap contribute to the temporal fluctuations in translation of single mRNAs. Collectively, Chapter 2 and 3 indicate that regulation of mRNA translation contributes to the proteome composition.
Chapter 4 and 5 illustrate how single-molecule imaging can be leveraged to investigate virus infection, replication, transcription, translation, and the interplay between a virus and a host cell. In Chapter 4, we establish a live-cell single-molecule imaging assay, called VIRIM, to examine translation and replication dynamics of +RNA viruses. VIRIM enables us to trace an infection from the first RNA molecule of the infecting viral particle and measure the timing, efficiency, and effect on the host cell of an infection. Specifically, we use VIRIM to identify the bottleneck of successful infection in the viral life cycle and study how, when, and how well the host cell’s antiviral response can try to attack this bottleneck.
Like +RNA viruses, the outcome of a -RNA virus infection depends on viral and host cell dynamics. In Chapter 5, we therefore expand the single-molecule virus imaging toolbox to also investigate -RNA viruses, particularly the Mononegavirales RSV. A combination of live-cell and fixed cell experiments reveals coordination and heterogeneity in expression of RSV genes. Furthermore, we observe that activation of the antiviral response anticorrelates with viral transcription, indicating that the heterogeneity in viral expression may have profound implications for the outcome of an infection.
Collectively, the new assays developed throughout this thesis have uncovered variations in dynamic processes and serve as a great starting point for many follow-up studies. Chapter 6 provides a summarizing discussion focusing on the limitations of the current tools and on potential future improvements and applications of the new assays.
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