Liquid-liquid phase separation (LLPS) represents a fundamental biophysical process in which a homogeneous solution of molecules spontaneously segregates into two or more distinct liquid phases, each enriched with different components. This phenomenon is of paramount importance in the context of cellular biology, as it enables the organization and regulation of biochemical processes without the need for membrane-bound organelles. LLPS thus serves as an essential tool for cellular compartmentalization, allowing cells to efficiently regulate and segregate various biochemical processes into different parts of the cell, through the formation of biomolecular condensates or membrane-less organelles. The focus of the presented doctoral thesis is on exploring and manipulating LLPS processes by designing and creating synthetic proteins based on coiled-coil (CC) building blocks. These designed proteins, consisting of segments of coiled-coils of varying lengths interconnected by different linkers and fluorescent proteins, enable visualization and precise monitoring of the dynamics of condensates. The work was based on a modular approach, allowing systematic variation in the strength and type of interactions between coiled-coils, directly affecting the properties of the resulting condensates, from their mobility and dynamics to their stability and rigidity. The experimental approach facilitated the development of a wide range of biomolecular condensates, including those composed of two or just one polypeptide chain. Particular attention was given to manipulating the strength of interaction between coiled-coils, enabling precise control over the transition between liquid, diffuse, and solid states of condensates. One of the key discoveries was the possibility of coexistence of multiple orthologous condensates within the same cell, opening doors to complex experimental setups and biological models. Additionally, strategies were developed for chemically induced regulation of the formation and disintegration of condensates, allowing precise temporal and spatial control over LLPS processes. The results of the doctoral thesis provide new insights into LLPS mechanisms and expand our understanding of the possibilities for manipulating cellular processes at the molecular level. This not only enhances our fundamental knowledge of biological systems but also opens promising avenues for applications in medicine, biotechnology, and synthetic biology, where designed biomolecular condensates could serve as platforms for drug delivery, cellular state sensors, or tools for reprogramming cellular behavior.
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