Cause pretty soon, smart cookies are thinking  – “What kind of device can I put together to make sure they all turn off by themselves…

                          

Specifically, Grecco et al. study the flow of information in the form of phosphate groups, a staple information tag that cells attach to their proteins to switch them on , off or give them special properties. Phosphate groups are attached to proteins by enzymes called Kinases, and removed by enzymes called Phosphatases. The fine balance between the kinase and phosphatase activities determines which proteins are phosphorylated and remain so for varying lengths of time.  A lot is known about which proteins get phosphorylated under what circumstances from tedious experiments over the last years that involve extracting the protoplasm into jumbled soup and then going about detecting how much phosphate is attached to which proteins. By replacing this step with a clever optical measurement, the authors opened the door to studying these interactions in living cells. Because the method is optical, and essentially works on a microscope (albeit not a very conventional one), the authors can not only tell which proteins get phosphorylated in response to a particular stimulus , but also where exactly in the cell these proteins are located.

To extract this information, the authors create a library of proteins containing phosphorylatable Tyrosine residues* , each tagged with one of the now famed fluorescent proteins. They adapted a technique called ‘reverse transfection’ to create a array of tiny spots of cells (a cellular array) , each expressing a different fluorescently-tagged protein. Then they added a stimulus – Epidermal Growth Factor, or EGF in short – an extracellular signal that sets off cellular signaling eventually leading to growth of a tissue. Then they added an antibody that would specifically recognize phosphorylated Tyrosines. The antibody itself was tagged with a particular fluorescent molecule. What they ended up with is cells, glowing with a fluorescent protein they had made it express, and an antibody that attached to this protein if its tyrosine were phosphorylated.

Now came the optical part. How does one find out if the antibody had bound to a protein? One of the most sensitive ways of detecting binding is to use a phenomenon called Foerster/Fluorescence Resonance Energy Transfer (FRET). Fluorescent molecules absorb light of a particular color, and electrons in them become ‘excited’, storing the energy for a brief instant. Eventually though, the electron gives off this energy as light again, but because some of the energy has been lost (no process is 100% efficient) , the light it gives is of a different color. Fluorescent molecules thus have an ‘absorption spectrum’ , and an ‘emission spectrum’. What happens , though, if there is another molecule sitting right next to this excited molecule that can absorb at exactly the same color that our excited molecule is about to emit? The laws of quantum physics say that , there is a chance that resonance will occur between these molecules and the excited molecule , instead of emitting light, will give away its energy to this other fluorescent molecule, that is perfectly suited to taking up this energy. This transfer can occur however , over only a very short distance , the so called Foerster radius – such that if it does occur, in almost all cases, it means that the two molecules are less than 20 angstroms from each other. In a solution of molecules, this is only likely to happen in any significant degree if the molecules are really bound to each other. In practice, it means the following, imagine a fluorophore that absorbs blue light and gives off green light. Another absorbs green light and gives off red light. If these molecules are so close as to be bound to each other, then shining blue light, will give off some green light, but also some red light. Some of the energy of the first fluorophore (the donor )has ‘leaked’ into the second one (the acceptor).

Left : A schematic representation of FRET-FLIM used for detecting Tyrosine Phosphorylation. Right : The high-throughput microcopy setup for measuring FLIM in biological samples.

FRET has been measured usually as the brightness of the signal. If energy is leaking between fluorophores, than the acceptor is brighter than it should be, and correspondingly the donor is dimmer than it should be. This method is not very precise in finding out how many of the donor-acceptor molecules are bound, though, since there are always unbound donors that confuse the signal. The authors use a different method to quantify FRET – they measure the average amount of time the donor fluorophores stay in their “excited” state – the fluorescence lifetime. If there is an acceptor bound and energy can leak, the donor molecules should remain in their excited state for a much shorter time – in other words their fluorescence lifetime will reduce. When performed on a microscope, the method is called Fluorescence Lifetime Imaging Microscopy (FLIM). The most useful part is that the average fluorescence lifetime reduces precisely in proportion to the amount of donors bound to acceptors, and FLIM can therefore tell us how much of a protein is phosporylated and thus bound to an antibody with an acceptor fluorophore.

The authors thus combined a high-throughput screening approach, with a sensitive optical method to detect protein phosphorylation, creating Cell Array – FLIM (CA-FLIM). They now had a way to see which of many proteins were phosphorylated, to what extent, and where in the cell, at the level of individual cells, when they added a stimulant. This is a level of detail in information that has been achieved in very few instances in biology. As with all cases of a data avalanche, however, the authors quickly found that biological variability was showing its effects – not all cells responded in the same way, and conventional methods of handling the data were incapable of resolving real phenomenon from the all-pervasive effects of this variability. So they had to develop a new way of analyzing this information, grouping cells into clusters based on how much of which protein was being phosphorylated. The mathematics of this new kind of ‘global analysis’ itself forms a significant proportion of the paper.

Finally, the authors compared their results with those obtained from the study of individual proteins done with tedious conventional methods. They found their data agreed with what other scientists had painstakingly discovered over years. Of course, CA-FLIM had revealed information of those proteins and many more in a matter of hours! What CA-FLIM added to the mix in many cases is information about the spatial aspects of this phosphorylation of proteins.

One point of critique here is that in order to detect the the phosphorylation of any protein, that protein needs to be expressed in its fluorescently tagged form. While this ectopic expression is relatively easy to do, it adds an extra population of that particular protein to what the cell itself is creating from its genome, known as the endogenous protein level. Since, the endogenous protein is not fluorescent, what happens to it remains in the dark. In all studies involving ectopic expression, the implicit assumption is that the fluorescent population behaves similarly to endogenous the non-fluorescent one , and all modifications occur to the same extent on both molecules. While this is more or less an reasonable assumption for many proteins , there is no way to be absolutely sure. Indeed, the assumption is in no way ironclad and several exceptions are known. In certain model organisms, such as yeast, it is possible to get around this problem by replacing the organism’s genes with fluorescently tagged versions of those same genes, but in mammalian cells, this is not yet technically possible.

CA-FLIM now adds to the growing list of methods that reduces the time required to detect cellular protein interactions by an order of magnitude. As technical challenges in implementation and automation are overcome, and CA-FLIM is expanded to other stimuli beyond the EGF used in this study, the multi-pronged nature of this approach should provide insights, or at least shine the light on interesting cellular phenomenon – increasing our knowledge of the basic unit of life further.

*Proteins can be phosphorylated on several amino acid residues , most commonly on Serine, Threonine and Tyrosine. Tyrosine phosphorylation seems more significant in the first steps of intracellular signaling and the authors focused on this particular kind.

NOTE : Nachiket Vartak , the author of this post , did not contribute to the study and development of CA-FLIM, but is affiliated to the same institution where the work was done. The author declares no conflict of interest.

Editor’s Selection IconGrecco, H., Roda-Navarro, P., Girod, A., Hou, J., Frahm, T., Truxius, D., Pepperkok, R., Squire, A., & Bastiaens, P. (2010). In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays Nature Methods, 7 (6), 467-472 DOI: 10.1038/nmeth.1458

                          

Disclaimer : The following is an excerpt from Salman Rushdie's novel "The Satanic Verses". This particular excerpt is , to my knowledge , in the public domain.

At the oasis of Yathrib the followers of the new faith of Submission found themselves landless, and therefore poor. For many years they financed themselves by acts of brigandage, attacking the rich camel-trains on their way to and from Jahilia.

Mahound had no time for scruples, Salman told Baal, no qualms about ends and means. The faithful lived by lawlessness, but in those years Mahound — or should one say the Archangel Gibreel? – should one say Al-Lah? – became obsessed by law. Amid the palm-trees of the oasis Gibreel appeared to the Prophet and found himself spouting rules, rules, rules, until the faithful could scarcely bear the prospect of any more revelation, Salman said, rules about every damn thing, if a man farts let him turn his face to the wind, a rule about which hand to use for the purpose of cleaning one's behind. It was as if no aspect of human existence was to be left unregulated, free.

The revelation – the "recitation" – told the faithful how much to eat, how deeply they should sleep, and which sexual positions had received divine sanction, so that they learned that sodomy and the missionary position were approved of by the archangel, whereas the forbidden postures included all those in which the female was on top. Gibreel further listed the permitted and forbidden subjects of conversation, and earmarked the parts of the body which could not be scratched no matter how unbearably they might itch. He vetoed the consumption of prawns, those bizarre other-worldly creatures which no member of the faithful had ever seen, and required animals to be killed slowly, by bleeding, so that by experiencing their deaths to the full they might arrive at an understanding of the meaning of their lives, for it is only at the moment of death that living creatures understand that life has been real, and not a sort of dream. And Gibreel the archangel specified the manner in which a man should be buried, and how his property should be divided, so that Salman the Persian got to wondering what manner of God this was that sounded so much like a businessman.

This was when he had the idea that destroyed his faith, because he recalled that of course Mahound himself had been a businessman, and a damned successful one at that, a person to whom organization and rules came naturally, so how excessively convenient it was that he should have come up with such a very businesslike archangel, who handed down the management decisions of this highly corporate, if non-corporeal, God.

After that Salman began to notice how useful and well timed the angel's revelations tended to be, so that when the faithful were disputing Mahound's views on any subject, from the possibility of space travel to the permanence of Hell, the angel would turn up with an answer, and he always supported Mahound, stating beyond any shadow of a doubt that it was impossible that a man should ever walk upon the moon, and being equally positive on the transient nature of damnation, even the most evil of doers would eventually be cleansed by hellfire and find their way into the perfumed gardens, Gulistan and Bostan. It would have been different, Salman complained to Baal, if Mahound took up his positions after receiving the revelation from Gibreel; but no, he just laid down the law and the angel would confirm it afterwards; so I began to get a bad smell in my nose, and I thought, this must be the odour of those fabled and legendary unclean creatures, what's their name, prawns.

The fishy smell began to obsess Salman, who was the most highly educated of Mahound's intimates owing to the superior educational system then on offer in Persia. On account of his scholastic advancement Salman was made Mahound's official scribe, so that it fell to him to write down the endlessly proliferating rules.

"All those revelations of convenience", he told Baal, "and the longer I did the job the worse it got. Anyway," Salman said near the bottom of the bottle, "finally I decided to test him."

"Little things at first. If Mahound recited a verse in which God was described as "all-hearing, all-knowing", I would write, "all-knowing, all-wise". Here's the point, Mahound did not notice the alterations. So there I was, actually writing the Book, or rewriting, anyway, polluting the word of God with my own profane language. But, good heavens, if my poor words could not be distinguished from the Revelation by God's own Messenger, then what did that mean? What did that say about the quality of the divine poetry?

Look, I swear, I was shaken to my soul. It's one thing to be a smart bastard and have half – suspicions about funny business, but it's quite another thing to find out that you're right. Listen, I changed my life for that man. I left my country, crossed the world, settled among people who thought me a slimy foreign coward for saving their, who never appreciated what I, but never mind that.

The truth is that what I expected when I made that first tiny change, "all-wise" instead of "all-hearing' – what I wanted -was to read it back to the Prophet, and he'd say, "What's the matter with you, Salman, are you going deaf? "And I'd say, "Oops, O God, bit of a slip, how could I", and correct myself.

But it didn't happen; and now I was writing the Revelation and nobody was noticing, and I didn't have the courage to own up. I was scared silly, I can tell you. Also, I was sadder than I have ever been. So I had to go on doing it. Maybe he'd just missed out once, I thought, anybody can make a mistake. So the next time I changed a bigger thing. He said "Christian", I wrote down "Jew".

He'd notice that, surely; how could he not? But when I read him the chapter he nodded and thanked me politely, and I went out of his tent with tears in my eyes. After that I knew my days in Yathrib were numbered; but I had to go on doing it. I had to. There is no bitterness like that of a man who finds out he has been believing in a ghost. I would fall, I knew, but he would fall with me. So I went on with my devilment, changing verses, until one day I read my lines to him and saw him frown and shake his head as if to clear his mind, and then nod his approval slowly, but with a little doubt. I knew I'd reached the edge, and that the next time I rewrote the Book he'd know everything. That night I lay awake, holding his fate in my hands as well as my own. If I allowed myself to be destroyed I could destroy him, too. I had to choose, on that awful night, whether I preferred death with revenge to life without anything. As you see, I chose, life. Before dawn I left Yathrib on my camel, and made my way, suffering numerous misadventures I shall not trouble to relate, back to Jahilia. And now Mahound is coming in triumph; so I shall lose my life after all. And his power has grown too great for me to unmake him now."

Baal asked, "Why are you sure he will kill you?"

Salman the Persian answered, "It's his Word against mine."

                          

This is just a quick post to warn people that Twitter messages are being used for phishing people's Twitter passwords.

I recieved a "Direct Message" on Twitter from @AccessDNA that went as follows : 

"hi. this you on here? http://blogger.djhxkcs.com " (Warning! Do not enter details on the linked page)


Of course, out of curiosity I visited the link , and waiting for me was a page that looked exactly like Twitter's Sign in page – except that it was posting the information to a different address , and would thus send any details I entered to some database. In time, I would expect my Twitter account to be usurped.

Watch out for suspicious messages and pay attention to the address, secure-connection icons and such, people.

                          

Locale : Muehlbachweg 13, Rottach-Egern, Germany