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Compositional Studies of Biomolecular Condensates

GFP-APEX2-PMLwt:proximity-based biotinylation reaction
GFP-APEX2-PMLwt:proximity-based biotinylation reaction

Biomolecular condensates are intracellular compartments found in eukaryotic cells. They are composed of concentrated proteins and nucleic acids and often play a significant role in maintaining cell homeostasis, similarly to other organelles (e.g. stress response, DNA repair, RNA metabolism). However, unlike canonical organelles, biomolecular condensates are not bounded by a membrane. Moreover, many biomolecular condensates behave as phase separated liquids in vivo, making them difficult to isolate and biochemically characterize. Some types of biomolecular condensates are known to be generally upregulated in certain types of cellular stresses (e.g. stress granules), while others are observed to accumulate proteins, which are later involved in disease progression (e.g. FUS in Amyotropic Lateral Sclerosis). To understand the mechanisms that bring the proteins and RNAs together to form biomolecular condensates and to be able to control their composition, we need to be able to first unravel their constituents. Therefore, having a way to study their composition, versatile to diverse types of biomolecular condensates is highly needed.

In my studies, I am using different biochemical and biophysical tools to analyze the composition of PML nuclear bodies and P bodies, two archetypal biomolecular condensates, in an unbiased, quantitative manner. To reach this goal, I am employing a proximity – based in situ biotinylation approach using the APEX2 enzyme.

In my initial experiments, I have tagged the PML protein and the Sp100 protein, two essential scaffold components of PML nuclear bodies, with APEX2 and I expressed them in HeLa cells. Upon treatment of these cells with biotin-phenol and a brief pulse of hydrogen peroxide, numerous proteins become selectively biotinylated. In the future, I will use mass spectrometry to identify the biotinylated proteins in both conditions, to generate an inventory of PML nuclear body resident proteins. This approach should be generally applicable to other biomolecular condensates.