Ghirlanda Lab

Disorderedly Functions

The Ghirlanda lab is working on designing functional, artificial membraneless organelles. In nature, membraneless organelles are subcellular structures formed by liquid-liquid phase separation (LLPS) and resemble liquid droplets suspended within the cellular medium. They exhibit rapid, dynamic self-assembly/disassembly in response to subtle environmental stimuli such as changes in the concentration of nucleic acids. The biological importance of these structures in physiological and diseased states is becoming increasingly clear, spurring extensive investigation of the mechanism of formation.

 

With our collaborators, Prof Sara Vaiana (Physics, ASU) and Prof. Matthias Heyden (SMS, ASU) we work on elucidating the mechanism of formation, characterizing the property of the droplets, and predicting the formation of LLPS droplets respectively. The hypothesis for ProteoCell is that LLPS can be exploited to generate designed membraneless organelles capable of performing complex functions. In the context of generating artificial cells, membraneless organelles could provide a technically simple, attractive compartmentalization strategy complementary to protein capsules and protein membrane.

 

The organelles are designed to specifically accommodate artificial metalloproteins that catalyze fuel production, by tuning the amino acid composition of the enzyme and of intrinsically disordered protein domains driving phase separation. Our long term goal is to compartmentalize complex “metabolisms” in order to increase the efficiency and specificity of reaction cascades and to regulate the formation of these “organelles” dynamically.

Liquid-Liquid_Phase_Separation.jpg

The Miracle of Phase Separation

IDPs are dynamic proteins that can change from a homogeneous mixture to a phase-separated solution. At separation, dense spheres of protein are formed. These droplets are the basis for our membraneless organelles.

The Unique Properties of IDPs

Ddx4, like many IDPs, has a unique amino acid composition. These proteins are generally populated by polar amino acids, most frequently serine and tyrosine. To increase flexibility, the sequence also contains a significant amount of glycine residues. This figure shows the composition of Ddx4 in comparison to the average proteins found in eukaryotes. Anything to the right of the dotted line is in higher concentration than average.

 Functionalizing Disorder 

In order to make a droplet a functioning organelle, it must include enzymes. Previous research has shown that enzymes by themselves will not enter the phase-separated droplet. In order to bring the enzymes inside, they are conjugated to the IDP through a linker.

Conjugation Map.jpg
GOx_HRP_Cascade.jpg

 The Big Picture 

An organelle is a complex system and as such, we must find ways to make our system complex. We propose using an enzyme cascade that will be encapsulated within the droplets. Systems can be as simple as two enzymes (horseradish peroxidase and glucose oxidase) or as complex as we wish. Each system will then be tied to a specific IDP to allow for distinct organelles.