Artificial Membraneless Organelle Carries Out Complex Biochemical Pathways

By Chengzi Guo ‘22

Caption: Microscopic image of coacacervated droplets
(Source: Springer-Verlag 2016)

Researchers at the Georgia Institute of Technology have recently created a novel synthetic organelle system made of localized membraneless solutions that can carry out biological processes. It has long been assumed that the compartmentalization of cells functions to create chemical environments that facilitate and regulate crucial biochemical reactions. This cellular compartmentalization often requires the physical separation of solutions by lipid membranes. Artificial cells mimic the conventional membrane-bounded organelles, which use a variety of lipids and steroids to partition the cell from its external environment.

Recently, scientists discovered the occurrence of membraneless compartmentalization through liquid-liquid phase separation (LLPS), which results in “pools” of distinct chemical solutions that do not mix with each other (2). One example of such “coacervation” is seen in the nucleolus, a multi-phase membraneless organelle that keeps ribonucleic acid (RNA), RNA polymerase, and pre-ribosomal particles in dedicated areas to facilitate transcription and translation (2). While membraneless organelles have been observed to store biomolecules, regulate enzyme kinetics, mediate stress, and offer protection from toxic molecules, the underlying mechanisms remain elusive.

Taking advantage of the varying pH, salt concentrations, constituent polymer concentrations, and the ionic strengths of different solutions, Kojima and his team created a layered system of droplets through LLPS, with the core being an ATP-PDDA (adenosine triphosphate and poly(diallyldimethylammonium chloride)) coacervate droplet. Furthermore, this droplet was capable of sequestering enzymes and reagents, an intermediate layer of dextran (DEX) droplet, and the outer polyethylene glycol (PEG) solution.

Multiple ATP-PDDA coacervates filled with different food colorings and dropped in the DEX-PEG system were able to retain their shape without diffusing for over 48 hours. Adding ions to specific droplets triggered the release of substances from the inner coacervate to the DEX layer, while maintaining the DEX-PEG boundary. This led to the food coloring mixing with each other but still remaining inside the larger DEX solution bubble. The results demonstrate that minute, yet distinctive, phase transitions along the interface of adjacent layers allow dynamic exchanges of molecules without the need of a lipid membrane.

Within cells, membraneless organelles facilitate signal transduction and metabolic cascade reactions by selective diffusion of certain molecules, while inhibiting movement of other molecules. Kojima’s team synthesized a similar enzymatic cascade beginning with glucose in the outer PEG solution being oxidized by glucose oxidase in DEX. The subsequent products of the reaction are shuttled into the ATP-PDDA coacervate, where another enzymatic reaction leads to the fluorescence of the core droplet. The system successfully demonstrates the conduction of cascading reactions across a series of spatial locations.

The team found that some enzymatic reactions are more efficient when the enzyme was partitioned away from the substrate through a phase-separated interface. Researchers had previously hypothesized higher enzymatic activity when substrate and enzyme are placed in the same compartment. The results reveal that increased substrate concentrations of DEX slowed DEX degradation by dextranase; this inhibition could be mitigated by localizing the enzyme to an ATP-PDDA droplet that is adjacent to, but phase-separated from, the DEX layer.

This membraneless system sheds light on new reaction acceleration mechanisms within cells through compartmentalization of enzyme from substrate. Kojima hopes that the research will enhance regulation of synthetic biological systems and provide clues to the origin of life.

References:

  1. Kojima, T., & Takayama, S. (2018). Membrane-less compartmentalization facilitates enzymatic cascade reactions and reduces substrate inhibition. ACS applied materials & interfaces.
  2. Georgia Institute of Technology. (2018, September 20). Synthetic organelle shows how tiny puddle-organs in our cells work: Lab model of membraneless organelle makes sugar metabolism reactions cascade. ScienceDaily. Retrieved October 1, 2018 from www.sciencedaily.com/releases/2018/09/180920175157.htm

 

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