Tag: packing

Posted on

Lifehack: How to pack two meters of chromatin into your cell’s nucleus, knot-free!

Original paper: The fractal globule as a model of chromatin architecture in the cell

The entirety of our genetic information is encoded in our DNA. In our cells, it wraps together with proteins to form a flexible fiber about 2 metres long known as chromatin. Despite its length, each cell in our body keeps a copy of our chromatin in its nucleus, which is only about 10 microns across. For scale, if the nucleus was the size of a basketball, its chromatin would be  about 90 miles long. How can it all fit in there? To make matters worse, the cell needs chromatin to be easily accessible for reading and copying, so it can’t be all tangled up. It’s not surprising then that scientists have been puzzled as to how this packing problem can be reliably solved in every cell. The solution is to pack the chromatin in a specific way, and research suggests that this may be in the form of a “fractal globule”. 

An equilibrium globule is the state that a polymer (a long repetitive molecular sequence, like chromatin) takes when it is left for a long time in a liquid that doesn’t dissolve it well. In such a liquid, the polymer is more attracted to itself than the molecules around it, so it collapses into a globule to minimize the amount of contact between itself and its surroundings. The resulting object is much denser than typical polymers in good solvents and is dense enough to fit inside a nucleus. However, like stuffing headphone cables into your pocket, it develops many knots and its different regions mix with one another.

On the other hand, if you change the polymer’s environment fast enough that it doesn’t have the time to fully equilibrate, then every piece of the polymer will locally collapse into its own globule. In other words, the polymer forms a globule made of smaller globules and is called a fractal globule. Fractals are objects which look the same at all scales, like the edge of a cloud or the coastline of England. If you zoom in or out on either of these objects, they look more or less the same. This isn’t an “equilibrium” state, meaning it will slowly fall out of this configuration. However, until the whole polymer equilibrates (which takes a long time), the chain has many desirable properties.

Figure 1. Simulated examples of fractal (A,C) and equilibrium globules (B,D), showing compartmentalization of different portions of the polymer. The chain color goes from red to blue as shown above. Compartmentalization means that parts of the chromatin stay near other parts with the same color (adapted from paper [1]).

We are interested in these globule states because they are dense enough that a globule of chromatin can fit inside of a cell nucleus. But it’s not enough to simply fit inside; the cell needs chromatin to avoid forming knots, since getting tangled would prevent the cell from properly reading its own DNA. Live cells also keep their chromatin nicely compartmentalized, that is, different regions along the genome stay spatially separated from one another. Unlike equilibrium globules, fractal globules have few knots and are also compartmentalized! To get a better picture for what this means, Leonid Mirny performed simulations of the different types of globules. Figure 1 shows the results of these simulations, highlighting how different the two states look in terms of knotting and separation of regions of the polymer. 

So it seems that the fractal globule state has all the properties we need for a good model of chromatin! But, as scientists, we know that no matter how well a theory fits the characteristics we want it to have, we need experimental evidence before believing anything. In the case of this fractal globule model for genome organization, evidence has come in the form of “contact probability maps”. These are collected from large populations of cells whose DNA is cut, spliced, and read in such a way that allows for a measurement of the probability that any two sites on the chromatin are touching at any given time. Among other things, these maps give us information about how chromatin is packed. So the question becomes, “what does the fractal globule model predict a contact probability map to look like?”

The fractal globule model doesn’t make exact predictions about where one will find specific segments of chromatin, but it does predict a contact probability as a function of distance between two sites, s. Specifically, the model predicts that the contact probability between two sites scales like 1/s. Meaning, if I look at sites that are twice as far apart along the polymer, then they are half as likely to be touching. This 1/s scaling is what was observed on intermediate scales (about 100,000 to 6 million base pairs) by looking at contact probability maps averaged over a whole population of cells.

We still don’t know how the cell maintains and tunes this fractal globule state, and we still have not developed a dynamic version of this picture, which is necessary since it is well-established that the chromatin in our cells is far from static. But this study gives us a new picture of how chromatin is organized inside cells. It isn’t randomly configured like headphone cables in your pocket or a ball of yarn. Rather it is folded onto itself in a self-similar way. This model is attractively simple, requires little fine-tuning, all while producing a long-lived state with segregated territories and easily accessible genes. 

[1] Mirny, Leonid (2011), The fractal globule as a model of chromatin architecture in the cell. Chromosome Res.

Featured image for the article is taken from Wikimedia Commons.

Posted on

Brick-by-brick to Build Tiny Capsules

Original paper: Colloidosomes: Selectively Permeable Capsules Composed of Colloidal Particles

Disclosure: The first author of the article discussed in this post, Anthony Dinsmore, is now my Ph.D. advisor. He did his postdoc at Harvard University a while ago, and consequently, I was never involved in this work.

In past two decades, several approaches have been developed and optimized to encapsulate a wide variety of materials, from food to cosmetics and the more demanding realm of therapeutic reagents. Inspired by biological cells, the first attempts were to use either natural or synthetic lipid molecules to form encapsulation vessels, the so-called liposomes. Then, with the increasing awareness of controlled release of cargo, especially for therapeutic purposes, advanced materials such as polymers were developed to form carrying vessels. There has been an enormous progress in encapsulation technologies, however, these methods can be limited in their applicability regarding encapsulation efficacy, permeability, mechanical strength, and for biological applications, compatibility. In this article, Anthony Dinsmore and his colleagues introduce a new platform and structure to encapsulate almost all types of materials with finely controlled and tuned properties.

Colloidosomes

An emulsion is produced typically by application of a shear force to a mixture of two or more immiscible liquids like the classical water-oil mixture. The resulting solution is a dispersion of droplets of one liquid in the other continuous liquid. In such case, an interface between the fluids exists that would impose an energy penalty on the system. Therefore, the system will always attempt to minimize it, in essence by reducing the area of the interface that is to merge the similar liquid droplets. Amphiphilic molecules are known to segregate in such interface to further reduce the energy and to inhibit the merging of droplets.  This segregation is not limited solely to molecules though. Solid particles tend to jam in the interface for the same reason to stabilize the emulsions. Inspired by the idea of particle-stabilized emulsions, which are known as Pickering emulsions, Dinsmore, and his colleagues have developed capsules made of solid particles. They adopt the name “Colloidosomes” by analogy to liposomes and demonstrate how the arrangement of these particles can be manipulated and controlled to achieve a versatile encapsulation platform.

Fabricating the Capsules

Colloidosomes are prepared first by making the emulsion in which the continuous phase contains the particles. For instance, in water-in-oil emulsions (“w/o”), water droplets become the core of the colloidosomes and particles are dispersed in the oil phase. Gentle agitation of such system results in particles being trapped in the water-oil interface (see Fig.1). The authors summarize the capsule formation in three main steps:

 

Screen Shot 2017-10-17 at 01.01.10
Fig 1. The colloidosome formation process is illustrated schematically in three steps. (A) a water/oil emulsion first is created through gentle agitation of the mixture for several seconds. (B) Particles are adsorbed to the w/o interface to minimize the total surface energy. Through sintering, van der Waals forces, and or addition of polycations ultimately the particles are locked in the interface. (C)In the end, the particle-stabilized droplet is transferred to water via centrifugation.

(a)  Trapping and stabilization. When the water-oil interface energy surpasses the difference between particle-oil and particle-water interface energy, particles are absorbed to the water-oil interface and become trapped due to the presence of a strong attractive well. This differs substantially from the case where particles were adsorbed to the interface via electrostatics, which requires the droplets to be oppositely charged to attract the particles. The packing of the particles at the interface is adjusted by controlling their interactions. Typically, the electrostatic interaction between particles, due to their surface chemistry, is utilized to stabilize the packing of the particle. For instance, in this study particles are coated with a stabilizing layer which in contact with water turns into a negatively charged layer.

 

(b)  Locking particles. To form an elastic and mechanically robust shell, the particles must be locked in the interface. This results in an intact capsule that can withstand mechanical forces. One way to obtain such elastic shell is to sinter the particles in place. Sintering is a thermally activated process in which the surface of particles melts and connects them to each other. Upon this local melting, the interstices among particles begin to shrink. With longer sintering times, it is possible to completely block the interstices, which results in very tough capsules with extremely high rupture points.  In this study, particles with 5 minutes of sintering yielded a 150 nm interstices size, and with 20 minutes, almost all the holes were blocked. By using particles with different melting temperatures, the sintering temperature can be adjusted over a wide range; this might be advantageous for encapsulants incompatible with elevated temperatures. Other ways of locking particles are electrostatic particle packing and packing via van der Waals forces. In the former case, for instance, a polyelectrolyte of opposite charge can be used to interact with several particles to lock them in place. In the latter case, for the van der Waals force to be effective, the steric repulsions and barrier must be destroyed so the surface molecules can get close enough for the London forces [1] to be strong.

 

After the Colloidosomes are formed, through gentle centrifuging, the fluid interface can be removed by exchanging the external fluid with one that is miscible with the liquid inside the colloidosome. In this step, having a robust shell to withstand shear forces crossing the water-oil interface is very important. This process ensures that the pores in the elastic shell control the permeability by allowing exchange by diffusion across the colloidosome shell. Now, with these steps and knowing parameters such as surface chemistry and locking condition, a promising system with characteristic permeability or cargo release strategies can be designed.

 

Tuning Capsule Properties; Permeation and Release

The most important feature of a colloidosome, as a promising encapsulant, is the versatility of permeation of the shell and or the release mechanisms. Sustained release can be obtained via passive diffusion of cargo via interstices that can be tuned via particle size and the locking procedure. With the mechanical properties of capsules optimized, shear forces can be used as an alternative release mechanism. For instance, minimally sintered polystyrene particles of 60 microns in diameter have shown to rupture in stresses that can be tuned by sintering time over a factor of 10. What makes the colloidosomes even more interesting is that one can choose different particles, with different chemistry, to have an auxiliary response, such as swelling, and dissolving of particular particles in response to the medium. It is also conceivable if one coats the colloidosome with the second layer of particles or polymers to improve or sophisticate the colloidosomes response. The latter can also mitigate the effects of any defect in the colloidosome lattice.

        With this unique platform, Dinsmore and colleagues stepped into the new realm of encapsulating materials of all kind. From therapeutic cargos to bioreactors, the chemical flexibility and even the ease of post-modification would expand the cargo type beyond molecules. For example, the authors show that living cells can be encapsulated in colloidosomes. Well, you may wonder, WHY? Imagine a protective shell around cells that keep them out of the reach of hostile microorganisms without compromising the cell’s vital activities such as nutrient trafficking and cell-to-cell crosstalk. 

[1]  London forces arise when the close proximity of two molecules polarizes both molecules. The resultant dipole work as a magnet to glue molecules together. Therefore, London forces are universal forces (and part of van der Waals forces), which takes effect when atoms or molecules are very close to each other.