Celestin N. Mudogo1,2 | Sven Falke1 | Hévila Brognaro1,3 | Michael Duszenko4 | Christian Betzel1

1Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg,Hamburg, Germany
2Department of Basic Sciences, School of Medicine, University of Kinshasa, Kinshasa,Democratic Republic of Congo
3Centre for Free-Electron-Laser Science, Hamburg, Germany
4Institute of Neurophysiology, University of Tübingen, Tübingen, Germany


Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have welldefined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities.
However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid-liquid, liquidgel, liquid-semi-crystalline, or liquid-crystalline phase separations. Liquid-liquid and liquid-gel MLOs form the majority of cellular phase separation events, while the occurrence of micro-sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. However, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known
today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization.


crowding agents, in vivo/in cellulo crystallization, liquid-liquid phase separation, membraneless and membrane-surrounded organelles