To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules.
Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. 4Integrated Biomedical Sciences Program, The University of Tennessee Science Center, Memphis, TN, United Statesīiomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response.Jude Children’s Research Hospital, Memphis, TN, United States 1Department of Structural Biology, St.Pounds 3, Khaled Khairy 2* and Richard Kriwacki 1,4* Baggett 1 †, Anna Medyukhina 2 †, Swarnendu Tripathi 1 †, Hazheen K.
0 kommentar(er)
