Speaker: Chiara Caldini (LENS) 

Cellular compartmentalization is the separation of the cellular interior into distinct compartments, each with specific conditions and materials, that enable the simultaneous execution of different metabolic processes. This organization is essential for regulating cellular functions, as it enhances efficiency by minimizing the loss of intermediate products. While the compartmentalization of eukaryotic cells is well understood, much less is known about the prokaryotic one. For a long time, prokaryotic cells, such as bacteria, were believed to lack subcellular organization, with biochemical reactions occurring in a disordered manner. However, recent studies on metabolic pathways suggest a certain degree of subcellular organization within bacteria too that would facilitate enzyme proximity to enhance reactions kinetics. Although molecular biology provides different techniques, such as two-hybrid systems, to analyse molecular interactions, imaging approaches are necessary to provide nanoscale spatial information about those interactions.

In our work we demonstrate how by combining Photo-Activated Localization Microscopy (PALM) with Expansion Microscopy (ExM) it is possible to perform three-dimensional simultaneous single-molecule co-localization and measure intermolecular distances with nanometres precision. The Ex-PALM approach is applied to study the spatial distribution of two enzymes, HisF and HisH, of the histidine metabolic pathway in Escherichia Coli bacteria whose interaction has already been demonstrated by previous studies. Using a plasmid-based transformation, each protein was tagged with a photoactivable fluorescent protein (HisF-PAGFP, HisH-PAmCherry) to enable single-molecule localization with nanometres accuracy. A negative control sample with two non-interacting enzymes, HisH and aaC, was also prepared to confirm our hypothesis that only interacting proteins co-localize.

To validate our theory, we first improved three-dimensional multicolour PALM imaging by reducing crosstalk and chromatic aberrations. This step was essential, as these aberrations are comparable in scale to the intermolecular distances of interest, and failing to correct them would compromise single-molecule localization accuracy, making it impossible to analyse densely packed subcellular structures, such as those found in bacteria. Next, we optimized the ExM protocol to achieve a 4.5-fold isotropic expansion of bacteria, enabling the study of bacterial subcellular organization. To prove the reliability of our analysis method, we applied it on both simulated and real data and found consistent results.