Green fluorescent protein and its fusion constructs induce intracellular puncta under defined conditions

Abstract Cells compartmentalize biomolecules using both membrane‑bound organelles and membrane‑less biomolecular condensates, the latter of which can be formed via liquid–liquid phase separation. Such condensates are linked to various physiological processes and disease progression; however, the evaluation of intracellular condensate formation remains challenging. Here, we examined the intracellular dynamics of three condensate-associated proteins, α-synuclein (αS), tau, and FUS, using fluorescent protein fusion constructs. Enhanced green fluorescent protein (EGFP)-fused αS and tau formed intracellular puncta upon serum removal, possibly due to elevated local protein concentrations. These puncta were sensitive to changes in cellular pH, divalent cation chelation, and the availability of metabolic substrates, and rapidly disappeared following exposure of cells to phosphate‑buffered saline. For tau–EGFP, tau-mediated microtubule binding competed with puncta formation. FUS–EGFP puncta were formed even under serum-containing conditions, exhibiting physicochemical properties distinct from those of αS–EGFP and tau–EGFP puncta. Thus, EGFP‑fused αS, tau, and FUS all formed puncta in cells. However, intracellular puncta were also observed in cells expressing EGFP without a fusion partner upon serum removal. These EGFP puncta exhibited properties similar to those of αS–EGFP and tau–EGFP puncta. Notably, the red fluorescent protein mRuby2 did not form puncta like EGFP, and serum removal failed to induce puncta formation by mRuby2‑fused αS and tau. Overall, our findings suggest that EGFP exhibits an intrinsic environment‑dependent propensity to form intracellular puncta, highlighting the importance of fluorescent protein choice and providing a basis for the reliable evaluation of condensate formation in living cells.