To elucidate the spatiotemporal control of gene expression, we are continuously developing single-molecule mRNA imaging and analysis tools for fungal cells and bacteria. These methods allow us to follow the entire mRNA life-cycle in single cells.

For fixed cells, we combine single-molecule fluorescent in situ hybridization (smFISH) with immunofluorescence (IF). This approach correlates, within the same cell, number and localization of mRNAs to protein expression.

To study dynamic mRNA expression in living cells, the best characterized method is the MS2-system. This approach uses stem loops from the MS2 bacteriophage (MS2 binding sites, MBS) inserted into the mRNA of interest, along with a plasmid encoded GFP-tagged MS2 capsid protein (MCP). This binds the stem loops and makes the mRNA fluorescent and detectable as single molecules using fluorescence imaging. We recently developed an improved MS2 system to accurately report mRNA stability and localization of unstable mRNAs.

An orthogonal stem-loop system was developed (PP7) and is used as a second colour for inter- or intramolecular labelling. Unlike biochemical approaches that result in ensemble measurements, these tools provide spatial and temporal information on individual molecular events, as well as on cell-to-cell variability.

Current efforts include:

• Multiplexing smFISH for the detection of tens to hundreds of mRNA species in single microbial cells.

• Development of smFISH protocols for non-canonical microorganisms (fungi and bacteria)

• Improving single molecule RNA imaging in living cells by using and developing optimized fluorescent reporters

• Development of imaging analysis algorithms to improve microbial cells segmentation and spots analysis in complex 3D structures.

The accurate tuning of cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression.

The recent development of high resolution imaging technologies to detect mRNAs and their translation state revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, providing the ability to tune gene expression locally. Therefore, mRNA trafficking and localization are a conserved and integral part of gene expression regulation.

In the lab, we study how the asymmetric localization of mRNA in fungi and bacteria contribute to optimal protein synthesis and cellular fitness.

Current efforts include:

• Studying bud mRNA localization and translation of cell cycle mRNAs, such as CLB2. We investigate how protein synthesis is regulated in the bud compartment.

• Elucidating how mRNAs are localized to subcellular compartments via specific RNA ZIP-codes.

• Characterization of mRNA localization in non-canonical microorganisms, such as filamentous fungi (pathogenic and non-pathogenic) as well as bacteria.

The formation of different cell types is a fundamental process during multicellular organisms development. Even unicellular species like yeast, undergo complex differentiation programs that allow them to increase the population diversity and to adapt to changing environments to improve their fitness in adverse conditions.

Both pathogenic (e.g. C. albicans) and non-pathogenic (e.g. S. cerevisiae) yeast undergo a reversible phenotypic switching in response to environmental changes (e.g. pH, temperature, lack of nutrients, quorum sensing). Under these conditions, fungi differentiate from single to filamentous cells called pseudohyphae or hyphae. Filamentous cells can further differentiate and form (poly)microbial biofilms, multicellular communities embedded in extracellular matrix where they are protected from sudden environmental changes

Here, we investigate fungal cells differentiation and biofilm formation using single-cell and single molecule approaches.

Current efforts include:

• Investigating the signalling pathways controlling fungal differentiation

• Elucidating the spatiotemporal control of fungal gene expression leading to

• Developing RNA and single-cell imaging technologies to provide fundamental insights on the spatial control of gene expression of a single cell organism that transitions to multicellularity.

The dynamic control of gene expression regulation lies at the basis of cellular adaptation. To respond to varying signals, genes are transcribed into mRNAs, transported in the cytoplasm and translated into proteins. Organization of these processes in space and time enables tuned protein expression. Genetically-identical cells behave differently, due to inevitable randomness of cellular processes. Thus, single cell approaches should be used to elucidate the spatial and temporal coordination of gene expression.

Here, we investigate how cells control mRNA levels to adjust protein expression, in particular during cell division and during rapid metabolic transitions. The field has largely focused on mRNA synthesis but less is known about the dynamic regulation of mRNA degradation.

Current efforts include:

• Investigating the degradation of cell cycle and metabolic genes

• Elucidating how the different step of mRNA metabolism, i.e. transcription, localization, translation and decay are coordinated

• We develop and use RNA imaging technologies to visualize the complete mRNA life cycle.