Determination of Intracellular Condensates by Increasing Scaffold Protein Concentration

The ability to perform liquid-liquid phase separation (LLPS) is a universal property of proteins and nucleic acids under specific conditions. However, many proteins may not be accessible to LLPS under physiological conditions, and only specific protein sequences appear to have the ability to phase separate under the conditions present in living cells. Great progress has been made in recent years in understanding the common molecular features of molecules, in which scaffold molecules are thought to be the drivers of phase separation, and molecules assigned to cohesions formed by scaffolds are referred to as clients. LLPS is a concentration-dependent process that occurs spontaneously only when the protein concentration reaches a critical threshold (called the saturation concentration C_sat). Further increases in concentration only lead to an increase in the volume of the density phase.

Fig. 1. Liquid-liquid phase separation is a function of concentration. (McSwiggen D T, et al., 2019)characterization. (Ozawa Y, et al., 2022)

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Protein concentration, especially scaffold proteins, is a key parameter in the formation of biomolecular cohesions and it controls the phase diagram of the underlying process. The key test to determine if LLPS occurs is to determine the critical concentration above which droplets are present and below which they are not. Therefore, CD BioSciences is committed to inducing intracellular LLPS by increasing the concentration of scaffolding proteins to further determine the material properties of condensates. Our experts develops the following two strategies to induce LLPS in vivo.

• Scaffold Protein Overexpression
  The extent of scaffold protein overexpression varies from case to case. We offer gene expression systems for mammalian studies allowing controllable induction of the protein overexpression, such as tetracycline-, cumate-, rapamycin-, light-, etc. Induced. In addition, we use three inducible promoters of increasing strength, including weak [GalS], medium [GalL], and strong [Gal1].

✓ This method is the most obvious and direct way to induce intracellular LLPS.

✓ This method is only applicable to the study of cohesive properties with known functional relevance.

• Subjugating Cells to Hyperosmotic Stress
  We indirectly affect intracellular target protein concentrations by subjugating cells to hyperosmotic stress in order to achieve C_sat values for LLPS induction. In this approach, changes in membrane ion channels, transporter protein activity, cytoskeletal remodeling, and water efflux in a hyperosmotic environment result in an increase in intracellular ionic strength and a decrease in cell volume, leading to an increase in intracellular crowding and scaffolding protein concentrations.

Our expert team carefully analyzes the data obtained using the overexpression method to induce LLPS. We perform quantitative measurements of scaffold proteins in the endogenous state and physiological abundance to analyze the causality and function of LLPS in vivo. Our service can provide guidance to improve consistency between in vitro and in vivo studies and to explore the role of scaffold proteins in LLPS. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.