![]() Depending upon the relative thermodynamic favorability of homotypic versus heterotypic interactions, LLPS causes the formation of cellular biomolecular condensates highly enriched in a single species or others containing multiple components ( Riback et al., 2020). These membrane-less compartments commonly form in cells through the process of liquid-liquid phase separation (LLPS), through which biomolecules exist within two distinct phases, a so-called “light phase” with low biomolecule concentrations and a “dense phase” wherein biomolecules are more highly concentrated ( Flory and Krigbaum, 1951 Banani et al., 2017). While many cellular compartments enclose their components within bilayer membranes, others lack a membrane and are referred to as membrane-less organelles or biomolecular condensates. The biomolecular components of cells are compartmentalized within organelles and other structures, wherein they perform their biological functions. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (K p) and Gibbs free energy of transfer (ΔG Tr), which quantify the favorability of a labeled biomolecule partitioning into puncta. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. 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* Richard Kriwacki 1,4* Baggett 1 † Anna Medyukhina 2 † Swarnendu Tripathi 1 † Hazheen K. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |