Kubrick And Cholesterol

Figure 1

You may wonder what the greatest filmmaker in history has anything to do with that greasy little molecule – apparently the only common aspect is that both have something to do with Private Gomer Pyle (Figure 1). Here, I want to discuss an interesting concept about the cell membrane – ‘membrane fluidity’, and how cholesterol interacts with the membrane in different ways.

Membrane fluidity is defined as the viscosity of the lipid bilayer in either a cell membrane or a synthetic membrane. This property is important because it affects the lateral diffusion of the proteins molecules within the membrane bilayer, which is something based on the fluid mosaic model of membrane structure.

Figure 2

At physiological condition, the presence of cholesterol will decrease the membrane fluidity and hence increase the stability of the membrane bilayer (Figure 2). Because the structure of cholesterol is a rigid steroid system, it will interfere with the lateral movement of the fatty acid tails on the phospholipid molecules, and the membrane is therefore stabilized as a result. Nevertheless, a certain level of dynamic interactions is still present in the physiological range.

Yet, when the temperature of the system changes, the story ceases to be simple. This is a relevant point because the whole idea of membrane fluidity not only works in living things, but also for systems generated in vitro. That means the temperature is not necessarily in the physiological range, and it will be interesting to see what cholesterol will do to the membrane bilayers at different temperatures.

Cholesterol is actually a bidirectional regulator for membrane fluidity. At low or high temperature, its presence on a membrane bilayer will lead to opposing effects. 

At low temperature, molecular motions are restricted and the phospholipid molecules have limited motion, thus they are 'frozen out' and appear in a solid-like phase on the membrane (or 'gel-phase' as some may put it). In this case, the introduction of cholesterol will increase the fluidity of the membrane, making the phospholipid molecules more mobile as a consequence.

Figure 3

Thus, cholesterol is a bit like Barry Lyndon from the Stanley Kubrick film here (Figure 3). Barry, an underdog from the 18th century, was a party-crasher to the aristocratic class in his era. These rich guys only conformed to the rules and did not want to stand out from the crowd so as not to lose the prestigious status, as if in a form of stasis. The introduction of Barry to the upper class would certainly destabilize that, as seen in Figure 3.

On the other hand, when temperature is high, there are a lot of molecular motions, and the phospholipids are moving a lot on the membrane and the bilayer assumes a fluid-like phase. The introduction of cholesterol will decrease the membrane fluidity and stabilize the membrane bilayer.

Figure 4

Now, cholesterol looks more like Sergeant Hartman from Full Metal Jacket (Figure 4). No matter those new recruits are hippies, fanboys of Dennis Hopper, or still on a trip of LSD, Sgt. Hartman would certainly to whip up some discipline from these kids, or gut-punch any of them to put them in the right place, in a geometry very much like a ‘bilayer’.

For all membrane systems, there is a characteristic transition temperature, where the bilayer will change from a gel-like consistency to the fluid-like consistency (Figure 5). The value depends on the exact chemical structure of the types of phospholipid in question.

Figure 5

One thing for sure, though - no matter there is another war or not, Ann Margaret will probably not come anyway!

by Ed Law

6/10/2018

#CellMembrane #MembraneFluidity #Cholesterol

Hold Me Tight, Laser Jock

Don't you have, uh, ray guns?

-The Terminator (1984)

The Nobel Prize winners for Physics this year have all contributed significantly to the physics of Lasers - 'Light Amplification by Stimulated Emission of Radiation'. Laser is not merely related to a 'B coefficient' from Einstein, it has moved beyond the theoretical field to contribute in many areas. The wonderful aspect is that the winners' contributions to laser have demonstrated applications in molecular biology, chemistry and medical science alike. 

Professor Arthur Ashkin is awarded the Nobel Prize for the invention of optical tweezers. That is a laser-based approach for trapping and manipulating particles. Optical tweezers rely on the radiation pressure of light (which generates force) to do the job. While it may appear weird or may sound like something from an episode of Star Trek, radiation pressure exists in all electromagnetic radiation. While this type of pressure appears weak for a human being, it certainly is not so for a particle. Ashkin has discovered that a particle interacting with a laser beam will be pushed towards a center with the highest intensity, due to the action of radiation pressure. By using a lens to properly focus the laser light,  the particle is further drawn towards the point with the greatest laser intensity. In a sense, it looks as if the laser beam is 'holding' the particle at that point. Ashkin's light trap will eventually be called the 'optical tweezers' as we know nowadays.

It is through the use of laser that optical tweezers can be devised and they have been applied in fields like biophysics and chemical biology, where single molecule or particle-sized materials have to be 'held up' and investigated.

Figure 1. From Ref. [1].

A famous use of optical tweezers in biochemistry is the study of molecular motors of the cytoskeleton system (Figure 1). Kinesin, a motor protein which is involved in moving cargo along the microtubules, has served as a testament to the use of optical tweezers in molecular biology.  

The kinesin protein is first attached to a bead which is held up by the optical tweezers. When the kinesin starts moving along the microtubules, the bead is pulled along and it can be visualized and quantitatively established how far the molecular motor has gone. In a sense, the displacement of the bead is like its escape from the clutch of the optical tweezers. At the point when the kinesin has moved too far away, the bead will then be 'bounced back' into the focus point of the optical tweezer - like a spring restoring to its equilibrium.

Figure 2. From Ref. [2].

The other half of the Nobel Prize was awarded to the approach known as 'chirped pulse amplification', which has led to the generation of high intensity and ultra-short optical pulses. In a nutshell, it is a stress-and-compress process. First, an ultra short laser pulse is stretched in time, reducing its peak power. The pulse is linearly chirped through a dispersion, and it is amplified in a laser material, afterwards it is compressed in time to its original duration. The result is an significant increase in peak power.As a result, the laser pulses have enhanced intensity. 

The high intensity laser is important because whenever we want to deal with situations requiring good precision, the energy source must be very focused to minimize undesirable thermal energy around the target, leading to collateral damage. That is indeed why high intensity laser has been used in surgery, because the ultra short pulse can give very precise cuts on the area.  

Ultra short laser pulses can also be applied in spectroscopy. For example, the generation of charges in semiconductor devices can be monitored by spectroscopic approaches, yet there are challenges associated with that. Because the time scale of the photophysical processes are very short, therefore we need fast and accurate spectroscopic techniques – known as transient absorption spectroscopy, to deal with that. A well-established approach is known as the pump-probe spectroscopy, which often uses laser as an energy source. From the name, we can realize there are 2 beams that leads to the process. A pump beam is used to excite the (semiconductors) material to the excited energy state; and a probe beam is used to monitor the excited state. The advantage of pump-probe is that because the pump and probe beam can be fine-tuned, through the pulse length of the laser and the photon density of the probe beam, therefore it can monitor processes with a reasonable wide time-scale, leading to its versatility as a tool for photophysics.

All in all, it is great to see that the contributions from physics can also be nicely applied in chemistry and biology too!

by Ed Law

Reference:

1. http://www.iiserpune.ac.in/~cathale/lects/2011monsoon/bio322-student/014.pdf

2. Zeitoun et. al., X-ray Chirped Pulse Amplification: towards GW Soft X-ray Lasers

Appl. Sci. 2013, 3(3), 581-592.

Release the Immuno-Brake

The Nobel Prize in Physiology or Medicine has just been announced. Professor James Allison and Professor Tasuku Honjo have won the Nobel Prize for their revolutionary contributions to an important understanding in immunology and its potential application in cancer therapy. The topic has led to my fond memories of taking immunology lectures in my undergraduate years. Here, I will share with you the Nobel Laureates' contribution to the understanding of 'negative immune regulation' and its inhibition to unleash the powers of immune cells.
 
The concept of the immune system is extremely complex because it entails a lot of molecular and biochemical events, so I will just describe the key processes here. The immune system defends us from various sort of microbes, because it has the ability to distinguish 'self' (the host’s body) from 'non-self' (the microbes). One of the most important players in the immune system is the T-lymphocytes (‘T-cells’). The T-cell is further categorized into cytotoxic (CD8) T cells and helper (CD4) T cells.

The CD4 T-cell is a like a midfielder in a soccer game, because upon its activation, it will lead to the downstream processes for the other components of the immune system, such as cytotoxic (CD8) T cells, to deal with and hence eliminate the enemy. The CD4 T cells achieve this end by engaging in the process of antigen presentation, where they 'present' a fragment of the broken-down microbe to an antigen-presenting cell (such as dendritic cells, macrophages and B lymphocytes). The resulting molecular interaction leads to an activated response, which serves as a signal for the other immune cells to take further action. Furthermore, a co-stimulation is often required to lead to a full and total immune response, and this is achieved by some activator proteins.

So, what's the deal with a nice guy helping us around to deal with the party-crashers? Well, if the immune system goes way too far off the map, the result can be catastrophic. Through genetic, environmental, or pathological factors, the immune system may over-react and in turn attack the host's healthy cells because it is misled to act in that way. The result is a variety of autoimmune diseases, and many of these can have drastic consequences for the host.

The immune system has been evolved throughout humanity to deal with this potential pitfall. Immunologists have discovered a number of inhibitory proteins, which serve as ‘brakes’ to balance out the action of the activator proteins. It is through this mechanism that a regulation is possible for the immune system, and this tight control is a theme in many biological systems.

(Top) Under normal circumstances, the CTLA-4 and PD-1 proteins will inhibit the T-cell response and serve as brakes to the immune system in 2 different mechanisms. CTLA-4 competes the active site with the activatory proteins, while PD-1 inhibits the T-cell response by binding to an alternative site on another (in some cases, cancerous) cells. PD-1, which stands for Programmed Cell Death 1 protein, presents a more complex scenario because while it can prevent autoimmune disease by suppressing the immune system from overacting in a normal body, it will cause problems in a cancerous case because the immune system is suppressed by its action. (Bottom) Anti-CTLA-4 and Anti-PD-1 are used to block the action of the two brake proteins, releasing the immune system to take care of the cancerous cells.
[Source: https://www.nobelprize.org/prizes/medicine/2018/press-release/ ]

The two Nobel Prize laureates discovered respectively two proteins that inhibit the T-cell response with slightly different mechanisms.  The proteins are CTLA-4 and PD-1 respectively. These two proteins are inhibitory proteins that prevent full T-cell activation and hence they can regulate the activities of the T-cell and the immune systems in the normal circumstances. Known as a ‘negative immune response’, the two proteins serve as ‘brakes’ that prevent the immune system from pressing the ‘go’ button in the wrong scenario.

What do these discoveries have anything to do with cancer therapy, then? Over the past century, researches have tried to find different approaches for cancer therapy. What is rather ironic is that our guardian angel - namely our immune system - has not been successful in the contribution to cancer therapy. Some researchers have even gone as far as adopting Edward Jenner's logic of vaccination to provoke a 'cancerous immune response', yet their efforts prove futile.

Professors Allison and Honjo have respectively developed similar insights to harness the negative immune regulation. Because if there is a way to stop the action of the brake (CTLA-4 and PD-1), then the immune system can be unleashed in full force to do the job, especially in the face of serious disease such as cancer. The solution is to use a specific antibody that can bind to and inactivate the ‘brake’ proteins. Thus, antibodies, known as anti-CLTA-4 and anti-PD-1, have been developed, and both research groups have successfully demonstrated that the antibodies can inhibit the ‘brake’ proteins. The immune system is released from the grip of the ‘brake’ proteins and thus can turn NBK on the cancer cells. (In fact, there is a type of immune cells called Natural Killer cells!). The two teams have successfully proved their ideas in some animal models, and the discovery has led to the development of ‘immune checkpoint therapy’.

The beauty of the discovery lies in the fact that, through the understanding of an important regulatory mechanism, a novel approach to cancer therapy can be devised and that will benefit cancer patients in the long term. The experiments the researchers have carried out to lead to this discovery also serve as a testament to the caliber of modern molecular biology, an intensely fascinating field on its own right.

by Ed Law
1/10/2018