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In Chapter 1, we explored the importance of building synthetic organelles, particularly in bacteria. From early efforts with fusion proteins and molecular scaffolds to creating more complex cellular structures, the idea of spatial organization in bacterial cells is a game-changer for biotechnology. However, attempts to construct membrane-bound organelles have been limited by our incomplete understanding of their biogenesis.
Fortunately, nature provides an exciting alternative to membrane-bound compartments - membraneless organelles. Let’s dive into the story of membraneless organelles, from their early discovery to modern efforts in understanding their formation, which guide us toward building synthetic versions.
Using the building metaphor, membrane-bound organelles are like rooms with walls, clearly separated from the rest of the space. In contrast, membraneless organelles are more like an open kitchen, condensed with food and materials. While they have no clearly defined barrier, they still block irrelevant elements from the outside and allow foods to move in and out as needed.
Many might think membraneless organelles are a "new discovery", especially given the attention they've received in recent years. In reality, the discovery of membraneless organelles goes back to the days of early microscopy.
Since Robert Hooke first observed and coined the term "cells," scientists in the 19th century began using optical microscopes to study cells. This work laid the foundation for the "cell theory", which became central to modern biology. Subcellular structures were identified and described early on around the same time:
(BTW, Cajal and Golgi shared a Nobel Prize in Physiology or Medicine in 1906 for research in the nervous system, which is another interesting story)
Now we know that some of them (e.g., nucleus, chloroplasts, mitochondria) are membrane-bound, while others (e.g., nucleolus, Cajal bodies) are membraneless.
However, during these early studies, the distinction did not exist, as the resolution of optical microscopes (200-500 nm)9 was too low to reveal membranes (~ 10 nm thickness)10.
It was generally accepted that some form of semi-permeable barrier existed around cells and possibly organelles, but the nature of this barrier remained unclear. It wasn’t until a series of pioneering experiments around 1925 that the lipid membrane nature of cell boundaries was suggested11, 12 (PS: from membraneless organelles, we will see boundary is actually not necessary for selective permeability).
With the use of electron microscopy in 1945, we finally saw the existence of cell membranes13.
At the same time, many organelles were found to be membrane-bound, and detailed structures were revealed. For instance, Porter et al discovered the lacy membrane structure of ER13, thus Porter coined the term "reticulum", meaning "network", which is used until now7. Since then, a membrane-centric understanding of cell models many of us were taught began to take place.
The discovery of membraneless organelles did not stop because of the dominance of membrane-based studies. In the late 20th century, scientists continued to observe structures that lacked membranes. During this time, they often refer to these membraneless entities as “granules”, because the term “organelle” was kinda "reserved" for membrane-bound structures. Examples include:
They also had many other names, such as "bodies", "subdomains", "speckles" et cetera. Only in recent years have they been classified as "membraneless organelles".
Speaking as a biologist, membrane-bound and -less organelles are essentially the same in functions. Both compartment tasks, making "breakrooms" in cells. For instance, you will find abstract structures in the processing of nascent RNA16 is very akin to the processing of polypeptides/proteins17, except that the former is membraneless, and the latter is membrane-bound.
The main difference lies in their physical nature. While the form and function of membrane-bound organelles are relatively straightforward, it wasn’t until recent years that the mechanism behind the formation of membraneless organelles - liquid-liquid phase separation (LLPS) - became clear.
So, what exactly is liquid-liquid phase separation (LLPS)?
To be clear, the term originates from physics, where "phase" refers to a distinct state of matter, like the difference between liquid water and solid ice.
In liquid-liquid phase separation, two liquids that were initially mixed begin to demix, separating into distinct phases, much like oil and water, or organic and aqueous (water) phases in liquid-liquid extraction. The concept has been studied for centuries, particularly in the context of colloids and emulsions18. These studies led to inventions like dishwasher detergents and laundry powders that emulsify oils and other stains. The term "phase separation" was officially used in the 1940s to describe the behavior of polymers and mixtures, first notably introduced in the paper by Flory in 1953, titled "Principles of Polymer Chemistry"19.
Despite its origins in physics and materials science, biologists have long been familiar with the concept. After all, many studies in colloids and emulsions were with biological materials. For example, milk is a classic emulsion of proteins dispersed in water, showing properties of both colloids and solutions, thus classified as hydrocolloids20. From a physics perspective, you could even consider membranes as a special case of liquid-liquid phase separation, where lipid molecules demix from water solutions into a specific bilayer structure.
During the late 19th and early 20th century, in studies of the physical properties of cells (notably, membranes were still invisible at the time), scientists saw cells as a kind of "jelly" or "colloid", and have noticed the phase-separating properties of intracellular structures. Key observations include:
These works laid the groundwork for understanding "phase" in biological contexts, where the cells were conceptualized as a disordered but dynamic system. Later, studies in this area are more about polymer sciences, and biologists' attention to intracellular spatial organization drifted towards membrane systems, which are more ordered, structured, and easier to understand, until.......
The tipping point was in 2009, when Anthony Hyman and Clifford Brangwynne demonstrated that P granules in C. elegans formed liquid-like droplets via LLPS25. Again, the fluid nature, the phase separation within cellular spaces were reconsidered.
Since membrane theories had dominated cell biology for decades, there was initial skepticism around LLPS since then. After all, under the right conditions, many proteins and RNAs can phase separate in vitro. How relevant is this to REAL biology?
However, the concept gradually became widely accepted as more examples of naturally occurring LLPS were discovered. In my opinion, the strongest evidence came from conserved membraneless organelles found in all eukaryotes, including:
Now the mechanism of LLPS has been well-studied. The LLPS process is driven by interactions between biopolymers. These are usually multi-valent interaction, such as oligomerization, binding among repeat units, or the condensation of intrinsically disordered regions (IDRs)26. In general, LLPS occurs when specific biomolecules - such as proteins and RNA - exceed a critical concentration and demix from their surrounding environment (cytoplasm or nucleoplasm) to form highly-concentrated liquid-like droplets. Given the process, a new terms "biomolecular condensate" is coined to refer to those membraneless organelles as well.
LLPS is sensitive to post-translational modifications, the physiochemical environment, and mutations. These liquid droplets can assemble and disassemble rapidly in response to cellular signals like stress or temperature changes. Mutations can lead to abnormal phase separation, causing diseases such as neurodegeneration.
These droplets are not random. They serve important functional purposes by compartmentalizing reactions without needing a membrane, but still maintaining selective permeability, actively exchanging materials and information with their surrounding environment. Complex structures could also occur, such as multi-layer droplets28, droplet assemblies29, and meshed networks30.
To appreciate why LLPS is ideal for building synthetic organelles, let’s first consider the limitations of membrane-bound structures. Constructing a membrane-bound organelle from scratch is extremely complex. Simply adding new lipid molecules to an existing membrane-bound structure requires numerous enzymes27. Building a vesicle from scratch is even more difficult, as the mechanism remains unclear to this day. Even if we manage to build a vesicle, we still need to add functional pores and receptors to enable selective permeability. The complexity is immense.
In contrast, LLPS offers several advantages for synthetic organelles:
In short, LLPS offers a straightforward, adaptable way to create de novo cellular compartments - an ideal platform for building synthetic organelles.
The discovery of LLPS has opened up a whole new frontier in synthetic biology. By mimicking nature’s membraneless organelles, we can build custom structures that are more dynamic, flexible, and efficient than traditional membrane-bound organelles. In fact, to study the properties of phase separation, many engineered phase-separating proteins have been designed to build model systems in vitro26.
The next step? Making it happen in vivo! In Chapter 3, we’ll dive into the design principles behind synthetic membraneless organelles, review successful designs to date, and explore what we can do to improve them in the future. Stay tuned!
Ailurus is a pioneering biocomputer company, programming biology as living smart devices, with products like PandaPure® that streamline protein expression and purification directly within cells, eliminating the need for columns or beads. Our mission is to make biology a general-purpose technology - easy to use and as accessible as modern computers.
