Like begets like. And so bags of membranes shed other bags of membranes. A cell during its course of its short (or long) tortuous life is expected to shed membranous structures. After all it is the primary container in which all life processes are carried. Does this process have a higher purpose is a question that had been pondered over and decided that it must be how the cells get rid of unwanted stuff. A few years ago, If I were offered to investigate these cellular garbage bags, my answer would have been an enthusiastic “Thanks but No thanks”.
(Image courtesy Johan Skog and Casey Maguire, Massachusetts General Hospital. I have met Johan, who is a wonderful person and is the CSO of exosome diagnostics.)
And hence I focused on disparate variety of things with no relations to the above, specifically on how neutrophils co-ordinate movement in complex tissue spaces. Neutrophils are the SWAT teams of the body. They are the first emergency responders that reach the site of a pathogen attack or injury to initiate a wide variety of inflammatory responses. Their modus operandi can be described right out of a scene of CSI. A police officer patrolling the highway smells a faint smell of smoke. He turns his head to detect the direction of the smoke and determines to be coming out of the wood. He decides to go into the wood to investigate, and in the meantime calls for a backup. He fears his backup wouldn’t know the direction he has taken into the woods and decides he should light up flares as he goes. As he approaches the scene of crime, shots ring out and he is fatally wounded. Before he dies, he shoots his flare gun into the air and calls for help from the backup that has arrived in wood’s edge. Seeing their comrade dead, the crack police team come out with guns blazing shooting down any one in their path.
Now replace the officer with neutrophil, smoke with bacterial peptides released by a pathogen, highway as the blood vessel, wood with interstitial tissue space, guns with the deadly neutrophil proteases. What was not known till a few years ago, is the nature of the flares or simply put how does neutrophil coordinate during subsequent neutrophil swarming during an inflammatory response. The answer turned out to be a rather unintuitive molecule called leukotriene B4 or LTB4. I call the molecule unintuitive not because it is not a potent molecule, it is one of prime targets in asthma medication, but because it is a lipid. And that is a problem. Let me illustrate.
In a perfect world, neutrophils sense a gradient of chemoattractant (e.g. Bacterial peptide) and move towards a region of higher concentration, a process that is called chemotaxis. In situation where the gradient is too shallow for the following cells to detect, it would release a second molecule which now can diffuse far and wide to recruit other neutrophil, hence increasing the range of response. This phenomenon was dubbed as signal relay. This second molecule was found to be LTB4. But being lipid it doesn’t diffuse well. It does not play very well with aqueous environment of the tissue and of course, it would not create a defined gradient (remember neutrophil can find its way by comparing differences or gradients of attractants in space). Unless of course there is something that facilitates in doing its job.
(from New paradigms in the establishment and maintenance of gradients during directed cell migration Current Opinion in Cell Biology, Volume 30, Pages 33-40 Ritankar Majumdar, Michael Sixt, Carole A Parent)
We started to find the facilitator by trying to locate where LTB4 was in the cell. We traced the location of the site of its active synthesis by mapping the proteins that were responsible for making this molecule. We found that these proteins were localized on atypical vesicular structures not found in a neutrophil’s natural repertoire. We took a very close look and found these were special bags-in-a-bag arrangement called multivesicular bodies. These are big bag that contains smaller bags which under certain conditions fuse with the cell membrane and release their content of smaller bags. These released smaller bags (or vesicles if you may) are called as exosomes. After painstaking ultrastructure sectioning of neutrophils migrating towards a bacterial tripeptide, we deciphered a series of events showing the genesis of the bag at the nuclear envelope to the fusion with the plasma membrane releasing smaller vesicles.
The rest of the story comprises of relatively boring monotonous sequence of validating the hypothesis by purifying exosomes, quantitating their content, knocking down the production of exosomes and their effects in chemotaxis etc. But one of the key insights came from a form of mixed cell experiments where we found cells talked to each other while they migrated. They talked through LTB4 by releasing them in exosomes. Without them the cells lose their sense of directionality. Without exosome, the dying police officers would have shot a very short feeble flare that the other police officers would have never seen. The exosomes acted as molecular beacons to the guardians of our body.
Very few engaged in biological research would believe exosomes to be the garbage bag of cells. They have been shown to be carrier of extracellular miRNA, they can transfer genetic material between cells, and even active proteins. They have been heralded as the best candidate for liquid biomarkers in cancer especially in glioblastomas where getting biopsies mean boring through your skull or spinal cord. Researchers who work with exosomes have an immensely difficult task of unravelling these microcosm of things, especially when one encounters the very fundamental question of “Did I just purify exosomes or some other stuff that the cells throws out?” The lack of standardization of markers and the tedious pain-in-the-butt purification process pose additional challenges. (It took ~ 500 ml of blood to purify ~ half a billion neutrophils to yield ~ 50-100 ug of pure exosomes)
All said, working on exosomes gives a feeling that a genome researcher would have felt in the last decade of the last millennia or the geneticist of the last century. The path to research is full of promise and is littered with red herrings, where paradigms change in matter of days and the rule of the game is that there is no rule. For me the moral of story is that One man’s junk is other man’s treasure. Literally.
(From Exosomes: secreted vesicles and intercellular communications, an excellent article by Clotilde Thery)
A few good reads and see.
One of my favorite video made by David Rogers in 1950s. This gives an idea of the persistence of a neutrophil chasing a bacterium. https://www.youtube.com/watch?v=I_xh-bkiv_c
An excellent review on leukocyte migration. For advanced readers I am afraid http://www.nature.com/nri/journal/v14/n4/full/nri3641.html
The finding of evidence of signal relay in neutrophils and how LTB4 plays a critical role. http://www.cell.com/action/showRelatedArticles?pii=S1534-5807%2812%2900084-6
The reason why I am writing this piece. “Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis.” http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002336
Richard Robinson has done a wonderful job of explaining the findings in the above paper in layman terms. “To Attract Others, Immune Cells Release a Packet Which Releases a Signal” http://journals.plos.org/plosbiology/article?id=info:doi/10.1371/journal.pbio.1002337.
An excellent review on the current standing and status of exosome research by Clotilde Thery. http://f1000.com/prime/reports/b/3/15
About the author: Ritankar Majumdar is a Post-Doctoral fellow at the National Cancer Institute, NIH. He has been trained as a biochemical engineer, although after completing his thesis on structure-function relationship of GPCRs from IISc, he refuses to identify himself as an one. He enjoys working on complex chemotactic systems, good music, decent food and living the fresh breathe of science.
This work by ClubSciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
